Compositions and methods for treating cancer

ABSTRACT

Provided herein are compounds used to inhibit the deamination enzyme responsible for the inactivation of therapeutic compounds, and methods of using them.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.12/755,106, filed Apr. 6, 2010, which claims priority to U.S.Provisional Application No. 61/167,112, filed Apr. 6, 2009. Thisapplication also is a continuation-in-part of U.S. application Ser. No.12/755,116, filed Apr. 6, 2010, which claims priority to U.S.Provisional Application No. 61/167,117, filed Apr. 6, 2009. Thisapplication also is a continuation-in-part of U.S. application Ser. No.12/755,122, filed Apr. 6, 2010, which claims priority to U.S.Provisional Application No. 61/167,119, filed Apr. 6, 2009. The entirecontent of each of these provisional and non-provisional applications isincorporated herein by reference.

BACKGROUND OF THE INVENTION

Cancer is the second most common cause of death in the U.S., exceededonly by heart disease, and accounts for 1 of every 4 deaths. Since 1990,in the U.S. alone, nearly five million lives have been lost to some formof cancer.

For example, breast cancer affects 186,000 women annually in the U.S.,and the mortality rate of this disease has remained unchanged for 50years. Surgical resection of the disease through radical mastectomy,modified radical mastectomy, or lumpectomy remains the mainstay oftreatment for this condition. Unfortunately, a high percentage of thosetreated with lumpectomy alone will develop a recurrence of the disease.

Lung cancer is the most common cause of cancer death in both sexes inthe United States. Lung cancer can result from a primary tumororiginating in the lung or a secondary tumor which has spread fromanother organ such as the bowel or breast. Primary lung cancer isdivided into three main types; small cell lung cancer; non-small celllung cancer; and mesothelioma. There are three types of non-small celllung cancer: squamous cell carcinoma, adenocarcinoma, and large cellcarcinoma. Mesothelioma is a rare type of cancer that affects thecovering of the lung called the pleura, and is often caused by exposureto asbestos.

Ovarian cancer accounts for about 3% of all cancers among women andranks second among gynecologic cancers, following cancer of the uterinecorpus. Ovarian cancer affects over 20,000 women in the United Stateseach year and causes some 15,000 deaths annually. If the disease isdiagnosed at the localized stage, the 5-year survival rate is over 90%;however, only about 19% of all cases are detected at this stage.

The incidence of pancreatic cancer has been increasing steadily in thepast twenty years in most industrialized countries, exhibiting thecharacteristics of a growing epidemiological problem.

Leukemia is a type of cancer that affects blood cells. Among thecurrently prescribed treatment regimes for leukemia are total bodyirradiation and chemotherapy. The two treatment regimes, however, pose aclinical dilemma: because leukemia is a cancer of the blood, all of thecells in the blood and all of the cells that arise in bone marrow mustbe treated in order to ensure destruction of the neoplastic cells.Destruction of all these cells leaves the patient in a severelyimmunodepressed state which could be as fatal as the leukemia.

Some cancer drugs are metabolized by an organism's naturally occurringenzymes such as adenosine deaminase (ADA, EC 3.5.4.4) and cytidinedeaminase (CDA, also termed cytosine nucleoside deaminase, cytidineaminohydrolase, or EC 3.5.4.5). These enzymes function to deaminatenatural aminopurine and aminopyrimidine nucleosides, respectively, inhuman and other organisms. These enzymes also convert activenucleoside-based cancer drugs into inactive metabolites. For example,the purine nucleoside drug arabinosyladenine (fludarabine, ara-A) isdeaminated by ADA; the resulting compound, with the parent amino groupreplaced with hydroxyl, is inactive as an antitumor agent compared tothe parent compound. Similarly, the antileukemia drug arabinosylcytosine(also termed cytarabine, Ara-C (or AraC);4-Amino-1-(β-D-arabinofuranosyl)-2(1H)-pyrimidinone; Cytosinearabinoside; or 1-(β-D-Arabinofuranosyl)cytosine) is metabolicallydegraded by CDA into inactive arabinosyluracil.

CDA is a component of the pyrimidine salvage pathway. It convertscytidine and deoxycytidine to uridine and deoxyuridine, respectively, byhydrolytic deamination (Arch. Biochem. Biophys. 1991, 290, 285-292;Methods Enzymol. 1978, 51, 401-407; Biochem, J. 1967, 104, 7P). It alsodeaminates a number of synthetic cytosine analogs which are clinicallyuseful drugs, such as ara-C mentioned above (Cancer Chemother.Pharmacol. 1998, 42, 373-378; Cancer Res. 1989, 49, 3015-3019; AntiviralChem. Chemother. 1990, 1, 255-262). Conversion of the cytosine compoundsto the uridine derivatives usually confers loss of therapeutic activityor addition of side-effects. It has also been shown that cancers thatacquire resistance to cytosine analog drugs often overexpress CDA (Leuk.Res. 1990, 14, 751-754). Leukemic cells expressing a high level of CDAcan manifest resistance to cytosine antimetabolites and thereby limitthe antineoplastic activity of such therapeutics (Biochem. Pharmacol.1993, 45, 1857-1861).

Tetrahydrouridine (THU, or1(β-D-Ribofuranosyl)-4-hydroxytetrahydropyrimidin-2(1H)-one) has beenknown as an inhibitor of cytidine deaminase for a number of years.

Various reports have suggested that co-administration with THU increasesthe efficacy and oral activity of cytidine-based drugs. For example, THUhas been shown to enhance the oral activity of anti-leukemic agent5-azacytidine (also termed AzaC; azacytidine; 5-azacitidine;azacitidine; 4-Amino-1-(β-D-ribofuranosyl)-1,3,5-triazin-2(1H)-one; or1-(β-D-Ribofuranosyl)-5-azacytosine) in L1210 leukemic mice (CancerChemotherapy Reports 1975, 59, 459-465). The combination of THU plus5-azacytidine has also been studied in a baboon sickle cell anemia model(Am. J. Hematol. 1985, 18, 283-288), and in human patients with sicklecell anemia in combination with orally administered 5-azacytidine (Blood1985, 66, 527-532).

THU has also been shown to enhance the oral efficacy of ara-C in L1210leukemic mice (Cancer Research 1970, 30, 2166; Cancer Invest 1987, 5,(4), 293-9), and in tumor-bearing mice (Cancer Treat. Rep. 1977, 61,1355-1364). The combination of intravenously-administered ara-C withintravenously-administered THU has been investigated in several clinicalstudies in humans (Cancer Treat. Rep. 1977, 61, 1347-1353; Cancer Treat.Rep. 1979, 63, 1245-1249; Cancer Res. 1988, 48, 1337-1342). Inparticular, combination studies in patients with acute myeloid leukemia(AML) and chronic myeloid leukemia (CML) have been performed (Leukemia1991, 5, 991-998; Cancer Chemother, Pharmacol. 1993, 31, 481-484).

Gemcitabine (also termed dFdC;1-(4-Amino-2-oxo-1H-pyrimidin-1-yl)-2-deoxy-2,2-difluoro-β-D-ribofuranose;or 2′-deoxy-2′,2′-difluorocytidine; or 2′,2′-difluoro-2′-deoxycytidine),another cytidine-based antineoplastic drug, has also been studied inconjunction with CDA inhibitors (Biochem. Pharmacol. 1993, 45,1857-1861). Co-administration with THU has been shown to alter thepharmacokinetics and bioavailability of gemcitabine in mice (Abstr.1556, 2007 AACR Annual Meeting, Apr. 14-18, 2007, Los Angeles, Calif.;Clin. Cancer Res. 2008, 14, 3529-3535).

5-Fluoro-2′-deoxycytidine (fluorocytidine, FdCyd) is anothercytidine-based anticancer drug which is an inhibitor of DNAmethyltransferase. The modulation of its metabolism and pharmacokineticsby THU in mice has been studied (Clin Cancer Res., 2006, 12, 7483-7491;Cancer Chemother. Pharm. 2008, 62, 363-368). FdCyd in combination withTHU is currently the subject of an ongoing clinical trial identified byNational Cancer Institute clinical trial no. NCT00378807.

5-Aza-2′-deoxycytidine (also termed decitabine; or, the active agent inthe branded product Dacogen®) is an antineoplastic agent for thetreatment of myelodysplastic syndrome (MDS), with potential utility forthe treatment of AML and CML as well. Like the other cytidine-baseddrugs, its oral bioavailability and efficacy are limited by deactivationby CDA. THU has been shown to improve the potency of decitabine in asickle cell disease model in baboons (Am. J. Hematol. 1985, 18,283-288). In addition, another known CDA inhibitor, zebularine, has beenshown to enhance the efficacy of decitabine in mice with L1210 leukemia(Anticancer Drugs 2005, 16, 301-308).

The results of the aforementioned studies suggest that there istherapeutic utility in the administration of CDA inhibitors togetherwith cytidine-based drugs such as decitabine, gemcitabine, ara-C,5-azacytidine and others. However, early CDA inhibitors such as THUsuffer from drawbacks that include acid instability (J. Med. Chem. 1986,29, 2351) and poor bioavailability (J. Clin. Pharmacol. 1978, 18, 259).

There is therefore an ongoing need for new, potent and therapeuticallyuseful inhibitors of CDA, and new compositions that are useful fortreating cancer or neoplastic disease.

SUMMARY OF THE INVENTION

There remains a need for new treatments and therapies for cancer andcancer-associated disorders. There is also a need for compounds usefulin the treatment or amelioration of one or more symptoms of cancer.Furthermore, there is a need for methods for inhibiting the activity ofthe enzyme cytidine deaminase.

Thus, provided herein are compounds of formula I, II, III, IV, V, VI,VII, or VIII. Also provided herein are pharmaceutical compositionscomprising (i) any one of the compounds of formula I, II, III, IV, V,VI, VII, or VIII and (ii) a pharmaceutically acceptable excipient or apharmaceutically acceptable carrier.

In another aspect, provided herein is a method of inhibiting cytidinedeaminase which comprises utilizing an effective amount of any compoundof the formulae I-VIII. In one embodiment of this method, the compoundis of the formula VIII.

In another aspect, provided herein is a pharmaceutical compositioncomprising a non-decitabine CDA substrate and any compound of theformulae I-VIII. In another aspect, provided herein is a pharmaceuticalcomposition comprising a non-decitabine CDA substrate and a compound offormula I. In still another aspect, provided herein is a pharmaceuticalcomposition comprising a non-decitabine CDA substrate and a compound offormula VIII. In certain embodiments of these pharmaceuticalcompositions, the non-decitabine CDA substrate may be cytidine,deoxycytidine, 5-azacytidine, gemcitabine, ara-C, tezacitabine,5-fluoro-2′-deoxycytidine, cytochlor, 5,6-dihydro-5-azacytidine,6-azacytidine, or 1-methyl-Ψ-isocytidine. In another embodiment, thenon-decitabine CDA substrate may be 5-azacytidine, gemcitabine, ara-C,tezacitabine, 5-fluoro-2′-deoxycytidine, or cytochlor. In anotherembodiment, the non-decitabine CDA substrate may be gemcitabine.

In another aspect, provided herein is a method of treating cancercomprising: administering to a subject a pharmaceutical compositioncomprising a non-decitabine CDA substrate; and administering to asubject a pharmaceutical composition comprising a compound of formula I.In one embodiment of this method, the non-decitabine CDA substrate maybe cytidine, deoxycytidine, 5-azacytidine, gemcitabine, ara-C,tezacitabine, 5-fluoro-2′-deoxycytidine, cytochlor,5,6-dihydro-5-azacytidine, 6-azacytidine, or 1-methyl-Ψ-isocytidine. Inanother embodiment, the non-decitabine CDA substrate may be5-azacytidine, gemcitabine, ara-C, tezacitabine,5-fluoro-2′-deoxycytidine, or cytochlor. In another embodiment, thenon-decitabine CDA substrate may be gemcitabine. In one embodiment ofthis method, the composition comprising a non-decitabine CDA substrateand the composition comprising a compound of formula I aresimultaneously administered. In another embodiment, the compositioncomprising a non-decitabine CDA substrate and the composition comprisinga compound of formula I are sequentially administered.

In another embodiment of this method, the cancer may be a hematologicalcancer or a solid cancer. Hematological cancers may be myelodysplasticsyndromes or leukemias. Leukemias may be acute myeloid leukemia orchronic myeloid leukemia. Solid cancers may be pancreatic cancer,ovarian cancer, peritoneal cancer, non small cell lung cancer, ormetastatic breast cancer.

In another aspect, provided herein is a method of treating cancercomprising:

administering to a subject a pharmaceutical composition comprising anon-decitabine CDA substrate; and administering to a subject apharmaceutical composition comprising a compound of formula VIII. Thenon-decitabine CDA substrate may be cytidine, deoxycytidine,5-azacytidine, gemcitabine, ara-C, tezacitabine,5-fluoro-2′-deoxycytidine, cytochlor, 5,6-dihydro-5-azacytidine,6-azacytidine, or 1-methyl-Ψ-isocytidine. In another embodiment, thenon-decitabine CDA substrate may be 5-azacytidine, gemcitabine, ara-C,tezacitabine, 5-fluoro-2′-deoxycytidine, or cytochlor. In anotherembodiment, the non-decitabine CDA substrate may be gemcitabine. In oneembodiment of this method, the composition comprising a non-decitabineCDA substrate and the composition comprising a compound of formula VIIIare simultaneously administered. In another embodiment, the compositioncomprising a non-decitabine CDA substrate and the composition comprisinga compound of formula VIII are sequentially administered.

In one embodiment of this method, the cancer may be a hematologicalcancer or a solid cancer. The hematological cancer may bemyelodysplastic syndromes or leukemias. Leukemias may be acute myeloidleukemias or chronic myeloid leukemias. Solid cancers may be pancreaticcancer, ovarian cancer, peritoneal cancer, non small cell lung cancer,or metastatic breast cancer.

In another aspect, the invention provides herein use of the compound offormula I for the manufacture of a medicament for treating cancer in asubject being treated with a non-decitabine CDA substrate. In stillanother aspect, the invention provides herein use of a compound offormula VIII for the manufacture of a medicament for treating cancer ina subject being treated with a non-decitabine CDA substrate. For eitherof these uses, the non-decitabine CDA substrate may be cytidine,deoxycytidine, 5-azacytidine, gemcitabine, ara-C, tezacitabine,5-fluoro-2′-deoxycytidine, cytochlor, 5,6-dihydro-5-azacytidine,6-azacytidine, or 1-methyl-Ψ-isocytidine. In another embodiment, thenon-decitabine CDA substrate may be 5-azacytidine, gemcitabine, ara-C,tezacitabine, 5-fluoro-2′-deoxycytidine, or cytochlor. In anotherembodiment, the non-decitabine CDA substrate may be gemcitabine. In oneembodiment of these uses, the cancer may be hematological cancers orsolid cancers. In still another embodiment of these uses, hematologicalcancers may be myelodysplastic syndromes or leukemias. The leukemia maybe acute myeloid leukemias or chronic myeloid leukemia. Solid cancersmay be pancreatic cancer, ovarian cancer, peritoneal cancer, non smallcell lung cancer, or metastatic breast cancer.

In another aspect, provided herein is a pharmaceutical compositioncomprising gemcitabine and a compound of formula I. In still anotheraspect, provided herein is a pharmaceutical composition comprisinggemcitabine and a compound of formula VIII.

In still another aspect, provided herein is a method of treating cancercomprising: administering to a subject a pharmaceutical compositioncomprising gemcitabine; and administering to a subject a pharmaceuticalcomposition comprising a compound of formula I. In one embodiment ofthis method, the composition comprising gemcitabine and the compositioncomprising a compound of formula I are simultaneously administered. Inanother embodiment of this method, the composition comprisinggemcitabine and the composition comprising a compound of formula I aresequentially administered.

In another aspect, provided herein is a method of treating cancercomprising: administering to a subject a pharmaceutical compositioncomprising gemcitabine; and administering to a subject a pharmaceuticalcomposition comprising a compound of formula VIII. In one embodiment ofthis method, the composition comprising gemcitabine and the compositioncomprising a compound of formula VIII are simultaneously administered.In another embodiment of this method, the composition comprisinggemcitabine and the composition comprising a compound of formula VIIIare sequentially administered.

In one embodiment of these methods, the cancer may be a hematologicalcancer or a solid cancer. The hematological cancer may be amyelodysplastic syndrome or a leukemia. The leukemia may be acutemyeloid leukemia or chronic myeloid leukemia. The solid cancer may bepancreatic cancer, ovarian cancer, peritoneal cancer, non small celllung cancer, or metastatic breast cancer.

In another aspect, provided herein is the use of a compound of formula Ifor the manufacture of a medicament for treating cancer in a subjectbeing treated with a composition comprising gemcitabine. In anotheraspect, provided herein is the use of a compound of formula VIII for themanufacture of a medicament for treating cancer in a subject beingtreated with a composition comprising gemcitabine. In one embodiment ofthese uses, the cancer may be a hematological cancer or a solid cancer.A hematological cancer may be a myelodysplastic syndrome or a leukemia.The leukemia may be acute myeloid leukemia and chronic myeloid leukemia.The solid cancer may be pancreatic cancer, ovarian cancer, peritonealcancer, non small cell lung cancer, or metastatic breast cancer.

In another aspect, provided herein is a method of inhibiting CDA frombinding a non-decitabine CDA substrate, which comprises utilizing aneffective amount of any compound of the formulae I-VIII. In oneembodiment of this method, the non-decitabine CDA substrate may becytidine, deoxycytidine, 5-azacytidine, gemcitabine, ara-C,tezacitabine, 5-fluoro-2′-deoxycytidine, cytochlor,5,6-dihydro-5-azacytidine, 6-azacytidine, or 1-methyl-Ψ-isocytidine. Inanother embodiment of this method, the compound is of the formula VIII,and the non-decitabine CDA substrate is gemcitabine.

In one embodiment, the present invention is directed to combinations of(i) any of the compounds given by formulae I-VIII and (ii) anon-decitabine CDA substrate. In another embodiment, the presentinvention is directed to pharmaceutical compositions comprisingcombinations of (i) any of the compounds given by formulae I-VIII, (ii)a non-decitabine CDA substrate, and (iii) a pharmaceutically acceptableexcipient. In yet another embodiment, the present invention is directedto methods of administering to a subject pharmaceutical compositionscomprising combinations of (i) any of the compounds given by formulaeI-VIII and (ii) a non-decitabine CDA substrate. In yet anotherembodiment, the present invention is directed to methods of treatingcancer comprising administering to a subject pharmaceutical compositionscomprising combinations of (i) any of the compounds given by formulaeI-VIII and (ii) a non-decitabine CDA substrate.

In a preferred embodiment, the present invention is directed tocombinations of (i) the compound given by formula VIII and (ii) anon-decitabine CDA substrate. In another preferred embodiment, thepresent invention is directed to pharmaceutical compositions comprisingcombinations of (i) the compound given by formula VIII, (ii) anon-decitabine CDA substrate, and (iii) a pharmaceutically acceptableexcipient. In yet another preferred embodiment, the present invention isdirected to methods of administering to a subject pharmaceuticalcompositions comprising combinations of (i) the compound given byformula VIII and (ii) a non-decitabine CDA substrate. In yet anotherpreferred embodiment, the present invention is directed to methods oftreating cancer comprising administering to a subject pharmaceuticalcompositions comprising combinations of (i) the compound given byformula VIII and (ii) a non-decitabine CDA substrate.

In another embodiment, the present invention is directed to combinationsof (i) any of the compounds given by formulae I-VIII and (ii) a CDAsubstrate; with the proviso that the CDA substrate is neither (a)decitabine, nor (b) a decitabine prodrug. In another embodiment, thepresent invention is directed to pharmaceutical compositions comprisingcombinations of (i) any of the compounds given by formulae I-VIII, (ii)a CDA substrate, and (iii) a pharmaceutically acceptable excipient; withthe proviso that the CDA substrate is neither (a) decitabine, nor (b) adecitabine prodrug. In yet another embodiment, the present invention isdirected to methods of administering to a subject pharmaceuticalcompositions comprising combinations of (i) any of the compounds givenby formulae I-VIII and (ii) a CDA substrate; with the proviso that theCDA substrate is neither (a) decitabine, nor (b) a decitabine prodrug.In yet another embodiment, the present invention is directed to methodsof treating cancer comprising administering to a subject pharmaceuticalcompositions comprising combinations of (i) any of the compounds givenby formulae I-VIII and (ii) a CDA substrate; with the proviso that theCDA substrate is neither (a) decitabine, nor (b) a decitabine prodrug.

In another preferred embodiment, the present invention is directed tocombinations of (i) the compound given by formula VIII and (ii) a CDAsubstrate; with the proviso that the CDA substrate is neither (a)decitabine, nor (b) a decitabine prodrug. In another preferredembodiment, the present invention is directed to pharmaceuticalcompositions comprising combinations of (i) the compound given byformula VIII, (ii) a CDA substrate, and (iii) a pharmaceuticallyacceptable excipient; with the proviso that the CDA substrate is neither(a) decitabine, nor (b) a decitabine prodrug. In yet another preferredembodiment, the present invention is directed to methods ofadministering to a subject pharmaceutical compositions comprisingcombinations of (i) the compound given by formula VIII and (ii) a CDAsubstrate; with the proviso that the CDA substrate is neither (a)decitabine, nor (b) a decitabine prodrug. In yet another preferredembodiment, the present invention is directed to methods of treatingcancer comprising administering to a subject pharmaceutical compositionscomprising combinations of (i) the compound given by formula VIII and(ii) a CDA substrate; with the proviso that the CDA substrate is neither(a) decitabine, nor (b) a decitabine prodrug.

In another embodiment, the present invention is directed to combinationsof (i) any of the compounds given by formulae I-VIII and (ii) a prodrugof a non-decitabine CDA substrate. In another embodiment, the presentinvention is directed to pharmaceutical compositions comprisingcombinations of (i) any of the compounds given by formulae I-VIII, (ii)a prodrug of a non-decitabine CDA substrate, and (iii) apharmaceutically acceptable excipient. In yet another embodiment, thepresent invention is directed to methods of administering to a subjectpharmaceutical compositions comprising combinations of (i) any of thecompounds given by formulae I-VIII and (ii) a prodrug of anon-decitabine CDA substrate. In yet another embodiment, the presentinvention is directed to methods of treating cancer comprisingadministering to a subject pharmaceutical compositions comprisingcombinations of (i) any of the compounds given by formulae I-VIII and(ii) a prodrug of a non-decitabine CDA substrate.

In another preferred embodiment, the present invention is directed tocombinations of (i) the compound given by formula VIII and (ii) aprodrug of a non-decitabine CDA substrate. In another preferredembodiment, the present invention is directed to pharmaceuticalcompositions comprising combinations of (i) the compound given byformula VIII, (ii) a prodrug of a non-decitabine CDA substrate, and(iii) a pharmaceutically acceptable excipient. In yet another preferredembodiment, the present invention is directed to methods ofadministering to a subject pharmaceutical compositions comprisingcombinations of (i) the compound given by formula VIII and (ii) aprodrug of a non-decitabine CDA substrate. In yet another preferredembodiment, the present invention is directed to methods of treatingcancer comprising administering to a subject pharmaceutical compositionscomprising combinations of (i) the compound given by formula VIII and(ii) a prodrug of a non-decitabine CDA substrate.

In another embodiment, the present invention is directed to combinationsof (i) any of the compounds given by formulae I-VIII and (ii) a prodrugof a CDA substrate; with the proviso that the prodrug of a CDA substrateis neither (a) decitabine, nor (b) a decitabine prodrug. In anotherembodiment, the present invention is directed to pharmaceuticalcompositions comprising combinations of (i) any of the compounds givenby formulae I-VIII, (ii) a prodrug of a CDA substrate, and (iii) apharmaceutically acceptable excipient; with the proviso that the prodrugof a CDA substrate is neither (a) decitabine, nor (b) a decitabineprodrug. In yet another embodiment, the present invention is directed tomethods of administering to a subject pharmaceutical compositionscomprising combinations of (i) any of the compounds given by formulaeI-VIII and (ii) a prodrug of a CDA substrate; with the proviso that theprodrug of a CDA substrate is neither (a) decitabine, nor (b) adecitabine prodrug. In yet another embodiment, the present invention isdirected to methods of treating cancer comprising administering to asubject pharmaceutical compositions comprising combinations of (i) anyof the compounds given by formulae I-VIII and (ii) a prodrug of a CDAsubstrate; with the proviso that the prodrug of a CDA substrate isneither (a) decitabine, nor (b) a decitabine prodrug.

In another embodiment, the present invention is directed to combinationsof (i) the compound given by formula VIII and (ii) a prodrug of a CDAsubstrate; with the proviso that the prodrug of a CDA substrate isneither (a) decitabine, nor (b) a decitabine prodrug. In anotherembodiment, the present invention is directed to pharmaceuticalcompositions comprising combinations of (i) the compound given byformula VIII, (ii) a prodrug of a CDA substrate, and (iii) apharmaceutically acceptable excipient; with the proviso that the prodrugof a CDA substrate is neither (a) decitabine, nor (b) a decitabineprodrug. In yet another embodiment, the present invention is directed tomethods of administering to a subject pharmaceutical compositionscomprising combinations of (i) the compound given by formula VIII and(ii) a prodrug of a CDA substrate; with the proviso that the prodrug ofa CDA substrate is neither (a) decitabine, nor (b) a decitabine prodrug.In yet another embodiment, the present invention is directed to methodsof treating cancer comprising administering to a subject pharmaceuticalcompositions comprising combinations of (i) the compound given byformula VIII and (ii) a prodrug of a CDA substrate; with the provisothat the prodrug of a CDA substrate is neither (a) decitabine, nor (b) adecitabine prodrug.

Provided herein are compositions comprising (i) decitabine and (ii) acompound of formula I, II, III, IV, V, VI, VII, or VIII, orpharmaceutically acceptable salts, C₁₋₆ alkyl esters, or C₂₋₆ alkenylesters thereof. In one aspect of the invention, provided herein arecompositions comprising (i) decitabine and (ii) a compound of formulaVIII, or pharmaceutically acceptable salts, C₁₋₆ alkyl esters, or C₂₋₆alkenyl esters thereof. In another aspect, provided herein is a methodof treating cancer comprising administering to a subject a compositioncomprising decitabine; and administering to a subject a compositioncomprising a compound of formula I, II, III, IV, V, VI, VII, or VIII, orpharmaceutically acceptable salts, C₁₋₆ alkyl esters, or C₂₋₆ alkenylesters thereof.

In another aspect, provided herein is a method of treating cancercomprising administering to a subject a composition comprisingdecitabine, and administering to a subject a composition comprising acompound of formula I, or pharmaceutically acceptable salts, C₁₋₆ alkylesters, or C₂₋₆ alkenyl esters thereof. In one embodiment of thismethod, the composition comprising decitabine and the compositioncomprising a compound of formula I are simultaneously administered. Inanother embodiment, the composition comprising decitabine and thecomposition comprising a compound of formula I are sequentiallyadministered.

In another aspect, provided herein is a method of treating cancercomprising administering to a subject a composition comprisingdecitabine; and administering to a subject a composition comprising acompound of formula VIII, or pharmaceutically acceptable salts, C₁₋₆alkyl esters, or C₂₋₆ alkenyl esters thereof. In one embodiment of thismethod, the composition comprising decitabine and the compositioncomprising a compound of formula VIII are simultaneously administered.In another embodiment, the composition comprising decitabine and thecomposition comprising a compound of formula VIII are sequentiallyadministered.

In another aspect, provided herein are pharmaceutical compositionscomprising (i) decitabine; (ii) a compound of formula I, II, III, IV, V,VI, VII, or VIII, or pharmaceutically acceptable salts, C₁₋₆ alkylesters, or C₂₋₆ alkenyl esters thereof; and (iii) a pharmaceuticallyacceptable excipient.

In another aspect, provided herein are pharmaceutical compositionscomprising (i) decitabine; (ii) a compound of formula VIII, orpharmaceutically acceptable salts, C₁₋₆ alkyl esters, or C₂₋₆ alkenylesters thereof; and (iii) a pharmaceutically acceptable excipient. Inany of these embodiments, the cancer may be a hematological cancer orsolid cancer. The hematological cancer may be myelodysplastic syndromeor leukemia. The leukemia may be acute myeloid leukemia or chronicmyeloid leukemia. The solid cancer may be pancreatic cancer, ovariancancer, peritoneal cancer, non small cell lung cancer, or metastaticbreast cancer.

In another aspect, provided herein is a method of treating cancercomprising administering to a subject a pharmaceutical compositioncomprising decitabine; and administering to a subject a pharmaceuticalcomposition comprising a compound of formula VIII, or a pharmaceuticallyacceptable salt, a C₁₋₆ alkyl ester, or a C₂₋₆ alkenyl ester thereof;and a pharmaceutically acceptable carrier.

In one embodiment, the cancer is a hematological cancer or solid cancer.The hematological cancer can be myelodysplastic syndrome or leukemia.The leukemia can be acute myeloid leukemia or chronic myeloid leukemia.The solid cancer can be pancreatic cancer, ovarian cancer, peritonealcancer, non small cell lung cancer, or metastatic breast cancer.

In yet another aspect, provided herein is a use of the compound offormula I, or a pharmaceutically acceptable salt, a C₁₋₆ alkyl ester, ora C₂₋₆ alkenyl ester thereof; for the manufacture of a medicament fortreating cancer in a subject being treated with a composition comprisingdecitabine.

In one embodiment, the cancer is a hematological cancer or solid cancer.The hematological cancer can be myelodysplastic syndrome or leukemia.The leukemia can be acute myeloid leukemia or chronic myeloid leukemia.The solid cancer can be pancreatic cancer, ovarian cancer, peritonealcancer, non small cell lung cancer, or metastatic breast cancer.

In still another aspect, provided herein is a use of the compound offormula VIII, or a pharmaceutically acceptable salt, a C₁₋₆ alkyl ester,or a C₂₋₆ alkenyl ester thereof; for the manufacture of a medicament fortreating cancer in a subject being treated with a composition comprisingdecitabine. In one embodiment, the cancer is a hematological cancer orsolid cancer. The hematological cancer can be myelodysplastic syndromeor leukemia. The leukemia can be acute myeloid leukemia or chronicmyeloid leukemia. The solid cancer can be pancreatic cancer, ovariancancer, peritoneal cancer, non small cell lung cancer, or metastaticbreast cancer.

In another aspect, provided herein is a method of preventing thedeamination of decitabine, which comprises utilizing an effective amountof any compound of the formulae I-VIII. In a particular embodiment ofthis method, the compound is a compound given by formula VIII.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a plot of total HPLC area-% purities of ER-876400(1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3,4-dihydro-1H-1,3-diazepin-2(7H)-one)as a function of time in simulated gastric fluid at 37° C.

FIG. 2 shows a plot of total HPLC area-% purities of ER-876437(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3,4-dihydro-1H-1,3-diazepin-2(7H)-one)as a function of time in simulated gastric fluid at 37° C.

FIG. 3 shows the effect of combining gemcitabine (1 mg/kg) PO andER-876437 (10 mg/kg) PO in the A2780 human ovarian cancer xenograftmodel.

FIG. 4 shows the UV spectrum of gemcitabine and ER-876437.

FIG. 5 shows HPLC chromatograms of gemcitabine in the presence of CDA inTris-HCl buffer at 37° C. at selected time points.

FIG. 6 shows HPLC chromatograms of gemcitabine in the presence of CDAand ER-876437 in Tris-HCl buffer at 37° C. at selected time points.

FIG. 7 shows the effect of ER-876437 on the levels of gemcitabine in thepresence of CDA in Tris-HCl buffer at 37° C.

FIG. 8 shows the UV spectrum of cytarabine and ER-876437.

FIG. 9 shows HPLC chromatograms of cytarabine in the presence of CDA inTris-HCl buffer at 37° C. at selected time points.

FIG. 10 shows HPLC chromatograms of cytarabine in the presence of CDAand ER-876437 in Tris-HCl buffer at 37° C. at selected time points.

FIG. 11 shows the effect of ER-876437 on the levels of cytarabine in thepresence of CDA in Tris-HCl buffer at 37° C.

FIG. 12 shows the effect of ER-876437 on the levels of cytarabine in thepresence of CDA in Tris-HCl buffer at 37° C.

FIG. 13 shows the UV Spectrum of decitabine and ER-876437.

FIG. 14 shows the HPLC chromatograms of decitabine in the presence ofCDA in Tris-HCl buffer at 37° C. at selected time points.

FIG. 15 shows the HPLC chromatograms of decitabine in the presence ofCDA and ER-876437 in Tris-HCl buffer at 37° C. at selected time points.

FIG. 16 shows the effect of ER-876437 on the levels of decitabine in thepresence of CDA in Tris-HCl buffer at 37° C.

DETAILED DESCRIPTION OF THE INVENTION

Enzymes that deaminate natural aminopurine and aminopyrimidinenucleosides can also convert active anti-cancer drugs into inactivecompounds in the human body. For example, the enzyme cytidine deaminasecan rapidly convert the amino group of certain drugs to a hydroxylgroup, rendering these compounds inactive. When an inhibitor of cytidinedeaminase is co-administered with a drug that is otherwise deaminated(and consequently deactivated) by this enzyme, improved anti-tumoractivity will be achieved.

The cytidine deaminase inhibitor(Z)-3,4-dihydro-1-((2R,3R,4S,5R)-tetrahydro-3,4-dihydroxy-5-(hydroxymethyl)furan-2-yl)-1H-1,3-diazepin-2(7H)-one(also referred to herein as “ER-876400”;1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3,4-dihydro-1H-1,3-diazepin-2(7H)-one;2H-1,3-Diazepin-2-one, 1,3,4,7-tetrahydro-1-β-D-ribofuranosyl-; or givenby chemical registry no. 75421-11-3) has been described in Liu, P. S. etal., J. Med. Chem. 24:662-666 (1981); and in U.S. Pat. No. 4,275,057(both of which are incorporated herein by reference in theirentireties). ER-876400 is given by formula IX:

(Here and elsewhere, where discrepancies exist between a compound's nameand a compound's structure, the chemical structure will control.)

Other cytidine deaminase inhibitors have previously been described ininternational application no. PCT/US2008/80163, filed on Oct. 16, 2008;in U.S. patent application Ser. No. 12/252,961, filed on Oct. 16, 2008;and in U.S. provisional patent application No. 60/980,397, filed Oct.16, 2007; all of which are hereby incorporated by reference in theirentireties.

Provided herein is a new class of inhibitors of cytidine deaminase(“CDA”). As described herein, these compounds have an improved half-lifeover other known compounds. In one embodiment, the compounds of theinvention have an improved half-life in simulated gastric fluid comparedto ER-876400. These compounds may be administered in combination withanother anti-cancer medicament (e.g., a non-decitabine CDA substrate ordecitabine) for purposes of treating cancer (e.g., myelodysplasticsyndrome, leukemia, pancreatic cancer, ovarian cancer, peritonealcancer, non small cell lung cancer, or metastatic breast cancer).

DEFINITIONS

The following definitions are used throughout this specification:

As used in the specification and claims, the singular forms “a,” “an,”and “the” include plural references unless the content clearly dictatesotherwise. Thus, for example, reference to a pharmaceutical compositioncomprising “a compound” may encompass two or more compounds.

The term “decitabine,” the active agent in the branded drug known asDACOGEN® or “5-aza-2′-deoxycytidine” refers to a compound having theformula:

“Alkyl” or “alkyl group” as used herein, means a straight-chain (i.e.,unbranched), branched, or cyclic hydrocarbon chain that is completelysaturated. Examples include without limitation methyl, ethyl, propyl,iso-propyl, butyl, iso-butyl, tert-butyl, n-pentyl and n-hexyl. In someembodiments, the alkyl chain is a C₁ to C₆ branched or unbranched carbonchain. In some embodiments, the alkyl chain is a C₂ to C₅ branched orunbranched carbon chain. In some embodiments, the alkyl chain is a C₁ toC₄ branched or unbranched carbon chain. In some embodiments, the alkylchain is a C₂ to C₄ branched or unbranched carbon chain. In someembodiments, the alkyl chain is a C₃ to C₅ branched or unbranched carbonchain. In some embodiments, the alkyl chain is a C₁ to C₂ carbon chain.In some embodiments, the alkyl chain is a C₂ to C₃ branched orunbranched carbon chain. “In certain embodiments, the term “alkyl” or“alkyl group” includes a cycloalkyl group, also known as a carbocycle.Exemplary C₁₋₃ alkyl groups include methyl, ethyl, propyl, isopropyl,and cyclopropyl.

“Alkenyl” or “alkenyl group,” as used herein, refers to a straight-chain(i.e., unbranched), branched, or cyclic hydrocarbon chain that has oneor more double bonds. Examples include without limitation ethenyl,propenyl, iso-propenyl, butenyl, iso-butenyl, tert-butenyl, n-pentenyland n-hexenyl. In some embodiments, the alkenyl chain is a C₂ to C₆branched or unbranched carbon chain. In some embodiments, the alkenylchain is a C₂ to C₅ branched or unbranched carbon chain. In someembodiments, the alkenyl chain is a C₂ to C₄ branched or unbranchedcarbon chain. In some embodiments, the alkenyl chain is a C₃ to C₅branched or unbranched carbon chain. According to another aspect, theterm alkenyl refers to a straight chain hydrocarbon having two doublebonds, also referred to as “diene.” In other embodiments, the term“alkenyl” or “alkenyl group” refers to a cycloalkenyl group.

“C₁₋₆ alkyl ester” refers to a C₁₋₆ alkyl ester where each C₁₋₆ alkylgroup is as defined above. Accordingly, a C₁₋₆ alkyl ester group of analcohol (—OH) has the formula —C(═O)O(C₁₋₆ alkyl), wherein the terminaloxygen occupies the position of the alcoholic oxygen.

“C₂₋₆ alkenyl ester” refers to a C₂₋₆ alkenyl ester where each C₂₋₆alkenyl group is as defined above. Accordingly, a C₂₋₆ alkenyl estergroup of an alcohol (—OH) has the formula —C(═O)O(C₂₋₆ alkenyl), whereinthe terminal oxygen occupies the position of the alcoholic oxygen.

Unless indicated otherwise, where a bivalent group is described by itschemical formula, including two terminal bond moieties indicated by “—,”it will be understood that the attachment is read from left to right.

Unless stereochemistry is depicted or otherwise stated or shown,structures depicted herein are also meant to include all enantiomeric,diastereomeric, and geometric (or conformational) forms of thestructure; for example, the R and S configurations for each asymmetriccenter, (Z) and (E) double bond isomers, and (Z) and (E) conformationalisomers. Therefore, single stereochemical isomers as well asenantiomeric, diastereomeric, and geometric (or conformational) mixturesof the present compounds are within the scope of the invention. Anytautomeric forms of the compounds of the invention are within the scopeof the invention.

Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures except for the replacement of hydrogen by deuteriumor tritium, or the replacement of a carbon by a ¹³C— or ¹⁴C-enrichedcarbon are within the scope of this invention. Such compounds areuseful, for example, as analytical tools or probes in biological assays.

“Treatment,” “treat,” and “treating” refer to reversing, alleviating,delaying the onset of, or inhibiting the progress of a disease ordisorder as described herein. In some embodiments, treatment may beadministered after one or more symptoms have developed. In otherembodiments, treatment may be administered in the absence of symptoms.For example, treatment may be administered to a susceptible individualprior to the onset of symptoms (e.g., in light of a history of symptomsor in light of genetic or other susceptibility factors, or in light of ahistory of symptoms and in light of genetic or other susceptibilityfactors). Treatment may also be continued after symptoms have resolved,for example to mitigate or delay their recurrence. “Treating” inreference to a disease, disorder or condition also refers to: (i)slowing a disease, disorder or condition, e.g., arresting itsdevelopment; or (ii) relieving a disease, disorder or condition, e.g.,causing regression of the clinical symptoms, or (iii) slowing a disease,disorder or condition and relieving a disease, disorder or condition.

“Preventing” in reference to a disease, disorder or condition refers topreventing a disease, disorder or condition, e.g., causing the clinicalsymptoms of the disease, disorder or condition not to develop.

“Inhibit,” “inhibitor,” and “inhibition” in reference to any of thecompounds given by formulae I-VIII (or the CDA inhibitors describedherein including without limitation any of their salts, alkyl esters oralkenyl esters) refers to reducing the ability of CDA to bind a CDAsubstrate, thereby reducing the ability of CDA to enzymaticallydeaminate a CDA substrate. Without being bound by any theory, acompound's ability to inhibit CDA may be due to the compound's abilityto bind the active site of a particular CDA protein thereby reducing theability of that particular CDA protein from binding a CDA substrate.“Inhibit,” “inhibitor,” and “inhibition” in this context does not referto a complete prevention of all CDA proteins from binding any CDAsubstrates. Rather, in this context, “inhibit,” “inhibitor,” and“inhibition” relate to the ability of CDA inhibitors to reduce theenzymatic deamination of CDA substrates by CDA. In one aspect, themethods of the present invention comprise contacting a cell with aneffective amount of a CDA inhibitor compound, i.e., a compound of theinvention, thereby inhibiting the activity of CDA.

“Patient” or “subject”, as used herein, means an animal subject,preferably a mammalian subject (e.g., dog, cat, horse, cow, sheep, goat,monkey, etc.), and particularly human subjects (including both male andfemale subjects, and including neonatal, infant, juvenile, adolescent,adult and geriatric subjects). “Subject” can also refer to a cell ortissue, in vitro or in vivo, of an animal or a human.

As discussed further below, the term “CDA substrate” refers to anycompound that may be deaminated by CDA. In one embodiment, the CDAsubstrate is decitabine. In one embodiment, the CDA substrate is neither(i) decitabine, nor (ii) a decitabine prodrug. The term “non-decitabineCDA substrate” as used herein refers to a CDA substrate that is neither(i) decitabine, nor (ii) a decitabine prodrug. The term “pro-drug of anon-decitabine CDA substrate” as used herein refers to a prodrug of aCDA substrate, wherein the CDA substrate is neither (i) decitabine, nor(ii) a decitabine prodrug. A “decitabine prodrug” is any compound thatis transformed in vivo into decitabine. Nonlimiting examples ofnon-decitabine CDA substrates include cytidine, deoxycytidine, aza-C(5-azacytidine), gemcitabine, ara-C (1-β-D-arabinofuranosylcytosine),tezacitabine, 5-fluoro-2′-deoxycytidine, cytochlor,5,6-dihydro-5-azacytidine, 6-azacytidine, and 1-methyl-Ψ-isocytidine.Cytidine and deoxycytidine are naturally occurring non-decitabine CDAsubstrates. In a particular embodiment, the non-decitabine CDA substrateis gemcitabine.

As discussed further below, a compound may be determined to be a CDAsubstrate through at least one of the following: (i) demonstration ofrelevant kinetics of deamination by CDA (K_(m)), and (ii) changes to itsexposure in a subject when administered with any one of the compoundsgiven by formulae I-VIII. A compound need not be positively evaluated byboth of these evaluations to be determined to be a CDA substrate.

A compound may be determined to be a CDA substrate by evaluation of itskinetics of deamination by CDA (K_(m)) using known assays. See, forexample, Bouffard, D. Y. et al., Biochem. Pharm. 45(9):1857-1861 (1993);Momparler, R. L. et al., Biochem. Pharm. 32(7):1327-1328 (1983);Cacciamani, T. et al., Arch. Biochem, Biophys. 290(2): 285-292 (1991);Wentworth, D. F. and Wolfenden, R., Biochemistry 14(23): 5099-5105(1975); and Vincenzetti, S. et al., Prot. Expression and Purification8:247-253 (1996), all of which are hereby incorporated by reference intheir entireties. The K_(m) value for cytidine was previously reportedas 12±0.9 μM; the K_(m) value for deoxycytidine was previously reportedas 19±4 μM. Chabot et al., Biochem. Pharm. 32(7):1327-8 (1983).Additionally, K_(m) values for ara-C (87±10 μM), gemcitabine (95.7±8.4and 5-azacytidine (216±51 μM) were also previously reported. Id. andBouffard, D. Y. et al., Biochem. Pharm. 45(9):1857-1861 (1993). TheK_(m) value for cytidine for CDA from human liver has also been reportedas 9.2 μM. Wentworth, D. F. and Wolfenden, R., Biochemistry 14(23):5099-5105 (1975). This publication also identifies the K_(m) value for5-azacytidine (58 μM) and for 6-azacytidine (4200 μM). Id.

Hence, CDA substrates include those compounds having a K_(m) value fromat least greater than 10 μM and up to 4500 μM. The K_(m) of the CDAsubstrate can fall within the range of 10 μM to 500 μM, from 10 μM to400 μM, from 10 μM to 300 μM, from 10 μM to 200 μM, from 10 μM to 175μM, from 10 μM to 150 or from 200 μM to 300 Alternatively, the K_(m)value is at least greater than 50 μM and no greater than 500 μM. TheK_(m) of the CDA substrate can fall within the range of 50 μM to 500 μM,from 50 μM to 400 μM, from 50 μM to 300 μM, from 50 μM to 200 μM, from50 μM to 175 μM, or from 50 μM to 150 μM.

A compound can also be determined to be a CDA substrate by evaluation ofcertain pharmacological parameters when administered to a subjectsimultaneously or sequentially with any one of the CDA inhibitors givenby formulae I-VIII. For example, a compound's exposure in a subject mayincrease when it is administered simultaneously or sequentially with oneof the CDA inhibitors given by formulae I-VIII. Such an evaluation wouldmeasure (i) the exposure of the compound when administered alone to asubject as compared to (ii) the exposure of the same compound whenadministered to a subject along with any one of the CDA inhibitors givenby formulae I-VIII. When simultaneous or sequential administration ofany one of the CDA inhibitors given by formulae I-VIII is found toincrease the exposure of the compound, then the compound is a CDAsubstrate.

The exposure of a compound may be followed by taking a biological samplefrom the subject (e.g., blood or urine) and evaluating the biologicalsample using analytical techniques (e.g., high pressure or highperformance liquid chromatography, or other analytical means). Theanalytical measurements may be used to determine the concentration-timeprofile of the compound and compute the compound's exposure using wellknown techniques. See, e.g., Gibaldi, M. and Perrier, D.,Pharmacokinetics, 2d ed., Marcel Dekker, New York, 1982, which is herebywholly incorporated by reference. Typically, the disappearance of thecompound is followed as a function of time.

Exposure experiments may be conducted for purposes of determiningwhether a substance is a CDA substrate regardless of the form of thesubstance (e.g., salts, polymorphs, prodrugs). Thus, for example, acompound may be administered to a subject as a prodrug in, for example,an esterified or other metabolizable protected form. Upon administrationto the subject, the prodrug may be, for example, de-esterified therebyreleasing the active drug in vivo. Whether this active drug is a CDAsubstrate may be determined by conducting the above described analyticalmeasurements with respect to the active drug. Alternatively, whether theprodrug itself is a CDA substrate may be determined by conducting theanalytical measurements described in the preceding two paragraphs withrespect to the prodrug.

A non-decitabine CDA substrate may be a drug used for treating a cancer;or, a drug used for treating any other disease or ailment.

As used herein, a “prodrug” is a composition that undergoes an in vivomodification when administered to a subject, wherein the product of thein vivo modification is a therapeutically effective compound. Prodrugsof compounds may be prepared by, for example, preparing a given compoundas an ester. The esterified form of the compound may be administered toa subject and may be de-esterified in vivo thereby releasing atherapeutically effective compound. Alternatively, some compounds may beprepared as prodrugs by adding short polypeptides (e.g., 1-6 aminoacids) to the compound. Such prodrugs when administered to a subject maybe cleaved (by, e.g., trypsin or other peptidases) thereby releasing atherapeutically effective compound. Formation of prodrugs is not limitedby the specific examples described herein. Other ways of preparingtherapeutically effective compounds as prodrugs are known. Examples ofprodrugs of non-decitabine CDA substrates include, without limitation,Gemcitabine elaidate (also termed 9(E)-Octadecenoic acid2′-deoxy-2′,2′-difluorocytidin-5′-yl ester;2′-Deoxy-2′,2′-difluoro-5′-O-[9(E)-octadecenoyl]cytidine; CP-4126; orCAS Registry no. 210829-30-4); Azelaic acid gemcitabine ester megluminesalt (also termed1-[5-O-(9-Carboxynonanoyl)-β-D-arabinofuranosyl]cytosine megluminesalt); other salts of Azelaic acid gemcitabine ester; and1-[4-(2-Propylpentanamido)-2-oxo-1H-pyrimidin-1-yl]-2-deoxy-2,2-difluoro-β-D-ribofuranose(also termed LY-2334737).

By the term “combination” is meant either a fixed combination in onedosage unit form, or a kit of parts for the combined administrationwhere a compound of the present invention and a combination partner maybe administered independently, at the same time, or separately withintime intervals that especially allow that the combination partners showa cooperative, e.g., additive or synergistic, effect, or any combinationthereof.

“Pharmaceutically acceptable” refers to those properties or substancesthat are acceptable to the patient from a pharmacological ortoxicological point of view, or to the manufacturing pharmaceuticalchemist from a physical or chemical point of view regarding composition,formulation, stability, patient acceptance, bioavailability andcompatibility with other ingredients.

“Pharmaceutically acceptable excipient” can mean any substance, notitself a therapeutic agent, used as a carrier, diluent, binder, orvehicle for delivery of a therapeutic agent to a subject, or added to apharmaceutical composition to improve its handling or storage propertiesor to permit or facilitate formation of a compound or composition into aunit dosage form for administration. Pharmaceutically acceptableexcipients are well known in the pharmaceutical arts and are described,for example, in Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa. (e.g., 20^(th) Ed., 2000), and Handbook ofPharmaceutical Excipients, American Pharmaceutical Association,Washington, D.C., (e.g., 1^(st) 2^(nd) and 3^(rd) Eds., 1986, 1994 and2000, respectively). Excipients may provide a variety of functions andmay be described as wetting agents, buffering agents, suspending agents,lubricating agents, emulsifiers, disintegrants, absorbents,preservatives, surfactants, colorants, flavorants, and sweeteners.Examples of pharmaceutically acceptable excipients include withoutlimitation (1) sugars, such as lactose, glucose and sucrose; (2)starches, such as corn starch and potato starch; (3) cellulose and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose,cellulose acetate, hydroxypropylmethylcellulose, andhydroxypropylcellulose; (4) powdered tragacanth; (5) malt; (6) gelatin;(7) talc; (8) excipients, such as cocoa butter and suppository waxes;(9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pHbuffered solutions; (21) polyesters, polycarbonates or polyanhydrides;and (22) other non-toxic compatible substances employed inpharmaceutical formulations.

“Pharmaceutically acceptable carrier” as used herein refers to anontoxic carrier or vehicle that does not destroy the pharmacologicalactivity of the compound with which it is formulated. Pharmaceuticallyacceptable carriers or vehicles that may be used in the compositions ofthis invention include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol,cyclodextrins, sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

“Pharmaceutically acceptable salt” refers to an acid or base salt of acompound of the invention, which salt possesses the desiredpharmacological activity and is neither biologically nor otherwiseundesirable. The salt may be formed with acids that include withoutlimitation acetate, adipate, alginate, aspartate, benzoate,benzenesulfonate, bisulfate butyrate, citrate, camphorate,camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,hemisulfate, heptanoate, hexanoate, hydrochloride hydrobromide,hydroiodide, 2-hydroxyethane-sulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate,thiocyanate, tosylate and undecanoate. Examples of a base salt includewithout limitation ammonium salts, alkali metal salts such as sodium andpotassium salts, alkaline earth metal salts such as calcium andmagnesium salts, salts with organic bases such as dicyclohexylaminesalts, N-methyl-D-glucamine, and salts with amino acids such as arginineand lysine. In some embodiments, the basic nitrogen-containing groupsmay be quarternized with agents including lower alkyl halides such asmethyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkylsulfates such as dimethyl, diethyl, dibutyl and diamyl sulfates; longchain halides such as decyl, lauryl, myristyl and stearyl chlorides,bromides and iodides; and aralkyl halides such as phenethyl bromides.

“Animal” refers to a living organism having sensation and the power ofvoluntary movement, and which requires for its existence oxygen andorganic food.

“Mammal” refers to a warm-blooded vertebrate animal with hair or fur.Examples include without limitation members of the human, equine,porcine, bovine, murine, canine or feline species.

“Cancer” refers to an abnormal growth of cells which tend to proliferatein an uncontrolled way and, in some cases, to metastasize (spread).Specific cancers types include without limitation the cancers identifiedin Publication No. US 2006/0014949 and the following:

-   -   cardiac: sarcoma (e.g., such as angiosarcoma, fibrosarcoma,        rhabdomyosarcoma, liposarcoma and the like), rhabdomyoma and        teratoma;    -   lung: bronchogenic carcinoma (e.g., such as squamous cell,        undifferentiated small cell, undifferentiated large cell,        adenocarcinoma and the like), alveolar (e.g., such as        bronchiolar) carcinoma, sarcoma, lymphoma, non-small cell lung        cancer and mesothelioma;    -   gastrointestinal: esophagus (e.g., such as squamous cell        carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma and the        like), stomach (e.g., such as carcinoma, lymphoma,        leiomyosarcoma and the like), pancreas (e.g., such as ductal        adenocarcinoma, insulinoma, carcinoid tumors, vipoma and the        like), small bowel (e.g., such as adenocarcinoma, lymphoma,        carcinoid tumors, Karposi's sarcoma, and the like), large bowel        (e.g., such as adenocarcinoma, and the like);    -   genitourinary tract: kidney (e.g., such as adenocarcinoma,        lymphoma, leukemia, and the like), bladder and urethra (e.g.,        such as squamous cell carcinoma, transitional cell carcinoma,        adenocarcinoma and the like), prostate (e.g., such as        adenocarcinoma, sarcoma), testis (e.g., such as seminoma,        teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma,        sarcoma, interstitial cell carcinoma, and the like);    -   liver: hepatoma (e.g., hepatocellular carcinoma and the like),        cholangiocarcinoma, hepatoblastoma, and angiosarcoma;    -   bone: osteogenic sarcoma (e.g., such as osteosarcoma and the        like), fibrosarcoma, malignant fibrous histiocytoma,        chondrosarcoma, Ewing's sarcoma, malignant lymphoma (e.g., such        as reticulum cell sarcoma), multiple myeloma, malignant giant        cell tumor chordoma (e.g., such as osteocartilaginous        exostoses), chondroblastoma, and giant cell tumors;    -   nervous system: skull, meninges (e.g., such as meningiosarcoma,        gliomatosis and the like), brain (e.g., such as astrocytoma,        medulloblastoma, glioma, ependymoma, germinoma [pinealoma],        glioblastoma multiform, oligodendroglioma, retinoblastoma,        congenital tumors and the like), spinal cord (e.g., such as        sarcoma and the like);    -   breast cancer;    -   gynecological: uterus (e.g., such as endometrial carcinoma and        the like), cervix (e.g., such as cervical carcinoma, and the        like), ovaries (e.g., such as ovarian carcinoma [serous        cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified        carcinoma], Sertoli-Leydig cell tumors, dysgerminoma, malignant        teratoma, and the like), vulva (e.g., such as squamous cell        carcinoma, intraepithelial carcinoma, adenocarcinoma,        fibrosarcoma, melanoma and the like), vagina (e.g., such as        clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma        (embryonal rhabdomyosarcoma], fallopian tubes (carcinoma) and        the like);    -   hematologic: blood (e.g., such as myeloid leukemia [acute and        chronic], acute lymphoblastic leukemia, chronic lymphocytic        leukemia, chronic myelocytic leukemia, myeloproliferative        diseases, multiple myeloma, myelodysplastic syndrome and the        like), Hodgkin's disease, non-Hodgkin's lymphoma;    -   skin: malignant melanoma, basal cell carcinoma, squamous cell        carcinoma, Karposi's sarcoma, and the like; and adrenal glands:        neuroblastoma.

As used herein, “therapeutically effective amount” refers to an amountsufficient to elicit the desired biological response. A therapeuticallyeffective amount of decitabine or gemcitabine, for example, is an amountsufficient to treat a disease or disorder as described herein. Atherapeutically effective amount of a compound given by formulae I-VIIIis an amount sufficient to increase the in vivo exposure of decitabineor a non-decitabine CDA substrate.

Throughout the specification, where discrepancies exist between thenamed compound and the structure shown, the structure shall control.Where any named synonyms (e.g., abbreviations, IUPAC names, generic orother chemic names, or registry numbers) provided for any particularcompound actually relate to different compounds, then the specificationshall be construed to refer to these compounds in the alternative.

Compounds of the Invention

The present invention provides compounds that inhibit the activity ofCDA. In another embodiment, these compounds may be administered incombination with another anti-cancer medicament (e.g., decitabine or anon-decitabine CDA substrate, a prodrug of a non-decitabine CDAsubstrate, or a precursor of a non-decitabine CDA substrate) forpurposes of treating cancer (e.g., myelodysplastic syndrome, acutemyelogenous leukemia, chronic myelocytic leukemia, non-small cell lungcancer, pancreatic cancer, ovarian cancer and breast cancer).

The present invention is directed to compounds of formula I:

wherein:

one of R₁ and R₂ is F, and the other is selected from H and F;

one of R₃ and R₄ is H, and the other is selected from H and OH;

where - - - - - - - is a covalent bond or absent, and R₄ is absent andR₃ is flat when - - - - - - - is a covalent bond;

or a pharmaceutically acceptable salt, a C₁₋₆ alkyl ester, or a C₂₋₆alkenyl ester thereof.

As used throughout the specification, the expression “R₃ is flat” meansthat R₃ resides in the same plane as the plane containing the carbon towhich R₃ is attached as well as the two carbon atoms immediatelyadjacent to the carbon to which R₃ is attached.

In one embodiment of formula I, R₁ and R₂ are each F.

In another embodiment, formula I is represented by a compound of formulaII:

or a pharmaceutically acceptable salt, a C₁₋₆ alkyl ester, or a C₂₋₆alkenyl ester thereof.

In another aspect, the present invention is directed to ER-876437 (or,2H-1,3-Diazepin-2-one,1,3,4,7-tetrahydro-1-β-(D-2-deoxy-2,2-difluororibofuranosyl)-; or1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyptetrahydrofuran-2-yl)-3,4-dihydro-1H-1,3-diazepin-2(7H)-one,shown as formula VIII). Here and elsewhere, where discrepancies existbetween a compound's chemical name and its structural depiction, thestructural depiction will control. Where discrepancies exist between thestructural depiction and ¹H NMR data, the ¹H NMR data will control.

In another aspect, the present invention is directed to compounds offormula VIII:

or a pharmaceutically acceptable salt, a C₁₋₆ alkyl ester, or a C₂₋₆alkenyl ester thereof.

In another aspect, the present invention is directed to compounds offormula VIII:

In another embodiment, formula I is represented by a compound of formulaIII:

wherein:one of R₃ and R₄ is H, and the other is selected from H and OH;or a pharmaceutically acceptable salt, a C₁₋₆ alkyl ester, or a C₂₋₆alkenyl ester thereof.

In another embodiment, formula I is represented by a compound of formulaIV:

or a pharmaceutically acceptable salt, a C₁₋₆ alkyl ester, or a C₂₋₆alkenyl ester thereof.

In one embodiment, formula IV is represented by a compound of formula V:

or a pharmaceutically acceptable salt, a C₁₋₆ alkyl ester, or a C₂₋₆alkenyl ester thereof.

In another embodiment, formula I is represented by a compound of formulaVI:

or a pharmaceutically acceptable salt, a C₁₋₆ alkyl ester, or a C₂₋₆alkenyl ester thereof.

In one embodiment, formula VI is represented by a compound of formulaVII:

or a pharmaceutically acceptable salt, a C₁₋₆ alkyl ester, or a C₂₋₆alkenyl ester thereof.

The present invention is also directed to pharmaceutical compositionscomprising a compound of formula I:

wherein:

one of R₁ and R₂ is F, and the other is selected from H and F;

one of R₃ and R₄ is H, and the other is selected from H and OH;

where - - - - - - - is a covalent bond or absent, and R₄ is absent andR₃ is flat when - - - - - - - is a covalent bond;

or a pharmaceutically acceptable salt, a C₁₋₆ alkyl ester, or a C₂₋₆alkenyl ester thereof; and a pharmaceutically acceptable carrier.

In another embodiment, the present invention is also directed topharmaceutical compositions comprising a compound of formula II:

wherein R3 is selected from H and OH; or a pharmaceutically acceptablesalt, a C₁₋₆ alkyl ester, or a C₂₋₆ alkenyl ester thereof; and apharmaceutically acceptable carrier.

In another embodiment, the present invention is also directed topharmaceutical compositions comprising a compound of formula III:

wherein:

one of R₃ and R₄ is H, and the other is selected from H and OH;

or a pharmaceutically acceptable salt, a C₁₋₆ alkyl ester, or a C₂₋₆alkenyl ester thereof; and a pharmaceutically acceptable carrier.

In another embodiment, the present invention is also directed to apharmaceutical composition comprising a non-decitabine CDA substrate anda compound of formula I:

wherein:one of R₁ and R₂ is F, and the other is selected from H and F;one of R₃ and R₄ is H, and the other is selected from H and OH;where - - - - - - - is a covalent bond or absent, and R₄ is absentwhen - - - - - - - is a covalent bond;or a pharmaceutically acceptable salt, a C₁₋₆ alkyl ester, or a C₂₋₆alkenyl ester thereof.

In one embodiment of the pharmaceutical composition comprising anon-decitabine CDA substrate and a compound of formula I, saidnon-decitabine CDA substrate is selected from the group consisting of5-azacytidine, gemcitabine, ara-C, tezacitabine,5-fluoro-2′-deoxycytidine, and cytochlor. In another embodiment, thepharmaceutical composition comprises a prodrug of a non-decitabine CDAsubstrate and a compound of formula I, said prodrug of a non-decitabineCDA substrate is selected from the group consisting of a prodrug of5-azacytidine, gemcitabine, ara-C, tezacitabine,5-fluoro-2′-deoxycytidine, or cytochlor.

In another embodiment, the present invention is also directed to apharmaceutical composition comprising a non-decitabine CDA substrate anda compound of formula VIII:

or a pharmaceutically acceptable salt, a C₁₋₆ alkyl ester, or a C₂₋₆alkenyl ester thereof.

In another embodiment, the present invention is also directed to apharmaceutical composition comprising a prodrug of a non-decitabine CDAsubstrate and a compound of formula VIII:

or a pharmaceutically acceptable salt, a C₁₋₆ alkyl ester, or a C₂₋₆alkenyl ester thereof.

In one embodiment of the pharmaceutical composition comprising anon-decitabine CDA substrate and a compound of formula VIII, saidnon-decitabine CDA substrate is selected from the group consisting of5-azacytidine, gemcitabine, ara-C, tezacitabine,5-fluoro-2′-deoxycytidine, and cytochlor. In another embodiment of thepharmaceutical composition comprising a prodrug of a non-decitabine CDAsubstrate and a compound of formula VIII, said prodrug of anon-decitabine CDA substrate is selected from the group consisting of aprodrug of 5-azacytidine, gemcitabine, ara-C, tezacitabine,5-fluoro-2′-deoxycytidine, and cytochlor.

In another embodiment of the invention, a pharmaceutical composition cancomprise (a) a compound of any one of formulae I-VIII and also (b) anon-decitabine CDA substrate. The non-decitabine CDA substrate may be5-azacytidine, gemcitabine, ara-C, tezacitabine,5-fluoro-2′-deoxycytidine, or cytochlor. In a particular embodiment, thepharmaceutical composition comprises (a) a compound of any one offormulae I-VIII and also (b) gemcitabine.

The present invention is directed to a pharmaceutical compositioncomprising decitabine and a compound of formula I:

wherein:

one of R₁ and R₂ is F, and the other is selected from H and F;

one of R₃ and R₄ is H, and the other is selected from H and OH;

where - - - - - - - is a covalent bond or absent, and R₄ is absent andR₃ is flat when - - - - - - - is a covalent bond;

or a pharmaceutically acceptable salt, a C₁₋₆ alkyl ester, or a C₂₋₆alkenyl ester thereof.

In one embodiment of formula I, R₁ and R₂ are each F.

In another embodiment, the present invention is directed to apharmaceutical composition comprising decitabine and a compound offormula II:

or a pharmaceutically acceptable salt, a C₁₋₆ alkyl ester, or a C₂₋₆alkenyl ester thereof.

In another aspect, the present invention is directed to a pharmaceuticalcomposition comprising decitabine and ER-876437 (or,2H-1,3-Diazepin-2-one,1,3,4,7-tetrahydro-1-β-(D-2-deoxy-2,2-difluororibofuranosyl)-; or1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3,4-dihydro-1H-1,3-diazepin-2(7H)-one,shown as formula VIII). Here and elsewhere, where discrepancies existbetween a compound's chemical name and its structural depiction, thestructural depiction will control. Where discrepancies exist between thestructural depiction and ¹H NMR data, the ¹H NMR data will control.

In another aspect, the present invention is directed to a pharmaceuticalcomposition comprising decitabine and a compound of formula VIII:

or a pharmaceutically acceptable salt, a C₁₋₆ alkyl ester, or a C₂₋₆alkenyl ester thereof.

In another aspect, the present invention is directed to a pharmaceuticalcomposition comprising decitabine and a compound of formula VIII:

In another embodiment, the present invention is directed to apharmaceutical composition comprising decitabine and a compound offormula III:

wherein:one of R₃ and R₄ is H, and the other is selected from H and OH;or a pharmaceutically acceptable salt, a C₁₋₆ alkyl ester, or a C₂₋₆alkenyl ester thereof.

In another embodiment, the present invention is directed to apharmaceutical composition comprising decitabine and a compound offormula IV:

or a pharmaceutically acceptable salt, a C₁₋₆ alkyl ester, or a C₂₋₆alkenyl ester thereof.

In one embodiment, the present invention is directed to a pharmaceuticalcomposition comprising decitabine and a compound of formula V:

or a pharmaceutically acceptable salt, a C₁₋₆ alkyl ester, or a C₂₋₆alkenyl ester thereof.

In another embodiment, the present invention is directed to apharmaceutical composition comprising decitabine and a compound offormula VI:

or a pharmaceutically acceptable salt, a C₁₋₆ alkyl ester, or a C₂₋₆alkenyl ester thereof.

In another embodiment, the present invention is directed to apharmaceutical composition comprising decitabine and a compound offormula VII:

or a pharmaceutically acceptable salt, a C₁₋₆ alkyl ester, or a C₂₋₆alkenyl ester thereof.

In another embodiment of the invention, a pharmaceutical composition cancomprise (a) a compound of any one of formulae I-VIII and also (b)decitabine.

Another embodiment of the invention is directed to methods ofadministering the pharmaceutical compositions described herein. Hence,the present invention is directed to a method of treating a subject forcancer comprising administering to the subject a non-decitabine CDAsubstrate; and administering to the subject a pharmaceutical compositioncomprising a compound of any one of formulae I-VIII. The non-decitabineCDA substrate and the compound given may be any one of formulae I-VIIIand may be administered to the subject sequentially or simultaneously. Asequential administration includes (a) first administering thenon-decitabine CDA substrate followed by (b) administering thepharmaceutical composition comprising a compound of any one of formulaeI-VIII. An alternative sequential administration includes (a) firstadministering the pharmaceutical composition comprising a compound ofany one of formulae I-VIII followed by (b) administering thenon-decitabine CDA substrate. A simultaneous administration includesadministering the non-decitabine CDA substrate and the pharmaceuticalcomposition comprising a compound of any one of formulae I-VIII at thesame time; or at substantially the same time.

When administration involves the separate administration (e.g.,sequential administration) of the first compound (e.g., a compound ofFormula I) and a second compound (e.g., a non-decitabine CDA substrate),as described herein, the compounds are administered sufficiently closein time to have the desired therapeutic effect. For example, the periodof time between each administration, which can result in the desiredtherapeutic effect, can range from minutes to hours to days and may bedetermined based on the properties of each compound such as potency,solubility, bioavailability, plasma half-life and kinetic profile. Forexample, the compounds may be administered in any order within 24-72hours of each other or within any time less than 24 hours of each other.Alternatively, the compounds may be administered in any order within oneweek of each other.

When the non-decitabine CDA substrate and the compound of any one offormulae I-VIII are administered sequentially, they are separatelyformulated and may be provided in any order. When the non-decitabine CDAsubstrate and the compound of any one of formulae I-VIII areadministered simultaneously, however, they may be either separatelyformulated or combined in the same formulation. When combined in thesame formulation, the non-decitabine CDA substrate and the compound ofany one of formulae I-VIII may be formulated so as to be released intothe subject at the same time or at different times. The release profileof a formulation comprising both the non-decitabine CDA substrate andthe compound of any one of formulae I-VIII includes the following:

-   -   A) release and bioavailability of the non-decitabine CDA        substrate followed by release and bioavailability of the        compound of any one of formulae I-VIII;    -   B) release and bioavailability of the compound of any one of        formulae I-VIII followed by release and bioavailability of the        non-decitabine CDA substrate;    -   C) release and bioavailability of the compound of any one of        formulae I-VIII at the same time as (or substantially at the        same time as) release and bioavailability of the non-decitabine        CDA substrate.

Thus, provided herein is a method of treating cancer, comprisingadministering to a subject in need thereof a composition comprising anon-decitabine CDA substrate and a compound of any one of formulae

When the non-decitabine CDA substrate is gemcitabine, the cancer to betreated may be colorectal cancer, pancreas tumor, breast tumor, braintumor, prostate tumor, lung tumor, metastatic or recurrentnasopharyngeal carcinoma, metastatic solid tumors, prostateadenocarcinoma, urinary tract tumor, renal tumor, renal cell carcinoma,transitional cell carcinoma, urethral cancer, head and neck tumor,nonresectable head and neck cancer, squamous cell carcinoma of the headand neck, malignant pleural or peritoneal mesothelioma, cervical cancer,uterus tumor, testis tumor, germ cell tumor, granulosa cell tumor of theovary, genital tract tumor, leukemia, adult T-cell lymphoma, B-celllymphoma, Hodgkins disease, lymphoproliferative disease, mantle celllymphoma, human myeloid and lymphoid leukemia, non-Hodgkin lymphoma,hematological cancers, cutaneous T-cell lymphoma, acute myelogenousleukemia, acute lymphoblastic leukemia hemotological neoplasm, chroniclymphocytic leukemia, sarcoma, leiomyosarcoma, soft tissue sarcomas,Kaposi's sarcoma, osteosarcoma of the bone, hepatobiliary system tumor,liver carcinoma, cholangiocarcinoma, gallbladder tumor, pancreaticductal adenocarcinoma, peritoneal tumor, intestine tumor, stomach tumor,endometrioid carcinoma, central nervous system tumor, small cell lungcancer, medulloblastoma, neuroblastoma or glioma.

In a particular embodiment, when the non-decitabine CDA substrate isgemcitabine, the cancer to be treated is pancreatic cancer, ovariancancer, metastatic breast cancer, non-small cell lung cancer, bladdercancer, transitional cell carcinoma, biliary tract cancer, urothelialcancer, gallbladder carcinoma, fallopian tube cancer, primary peritonealcancer, squamous cell carcinoma of the head and neck, hepatocellularcarcinoma, liver tumor, lung carcinoma, uterine cervix tumor or coloncancer.

In still another embodiment, when the non-decitabine non-decitabine CDAsubstrate is gemcitabine, the cancer to be treated is non-small celllung cancer, pancreatic cancer, bladder cancer, breast cancer, oroesophageal cancer. Thus, provided herein is a method of treatingnon-small cell lung cancer, pancreatic cancer, bladder cancer, breastcancer, or oesophageal cancer in a subject in need thereof, comprisingadministering to the subject a pharmaceutical composition comprising acompound of Formula VIII and gemcitabine.

In another embodiment, provided herein is a method of treating cancer ina subject in need thereof, comprising administering to the subject acomposition comprising gemcitabine and ER-876437. In still anotherembodiment, provided herein is a method of treating cancer in a subjectin need thereof, comprising administering to the subject a compositioncomprising gemcitabine and ER-876437, wherein the cancer is selectedfrom the group consisting of non-small cell lung cancer, pancreaticcancer, ovarian cancer and breast cancer.

In another embodiment, provided herein is a method of treating psoriasisvulgaris, smallpox, liver cirrhosis, thromboembolism, meningitis,salivary gland disease, urethral disease, lymphoproliferative disease,or neutropenia in a subject in need thereof, comprising administering tothe subject a composition comprising gemcitabine and ER-876437.

In another embodiment, the invention is directed to combinations of anyone of the compounds given by formulae I-VIII with a prodrug of anon-decitabine CDA substrate. Such combinations may be formulated oradministered in all manners as described herein for combinationscomprising the non-decitabine CDA substrate.

In another embodiment of the invention, the non-decitabine CDA substrateand the compound of any one of formulae I-VIII may be administeredsequentially (in any order) or simultaneously with other pharmaceuticalagents typically administered to subjects being treated for cancer. Suchother pharmaceutical agents include without limitation anti-emetics,agents that increase appetite, other cytotoxic or chemotherapeuticagents, and agents that relieve pain. The non-decitabine CDA substrateand the compound of any one of formulae I-VIII may be formulatedtogether with or separately from such other pharmaceutical agents.

A combination with such other pharmaceutical agents can either result insynergistic increase in anti-cancer activity, or such an increase may beadditive. Compositions described herein typically include lower dosagesof each compound in a composition, thereby avoiding adverse interactionsbetween compounds or harmful side effects, such as ones which have beenreported for similar compounds. Furthermore, normal amounts of eachcompound when given in combination could provide for greater efficacy insubjects who are either unresponsive or minimally responsive to eachcompound when used alone.

A synergistic effect may be calculated, for example, using suitablemethods such as the Sigmoid-Emax equation (Holford, N. H. G. andScheiner, L. B., Clin. Pharmacokinet. 6: 429-453 (1981)), the equationof Loewe additivity (Loewe, S, and Muischnek, H., Arch, Exp. Pathol.Pharmacol. 114: 313-326 (1926)) and the median-effect equation (Chou, T.C. and Talalay, P., Adv. Enzyme Regul. 22: 27-55 (1984)). Each equationreferred to above may be applied to experimental data to generate acorresponding graph to aid in assessing the effects of the drugcombination. The corresponding graphs associated with the equationsreferred to above are the concentration-effect curve, isobologram curveand combination index curve, respectively.

Another embodiment of the invention is directed to methods ofadministering the pharmaceutical compositions described herein. Hence,the present invention is directed to a method of treating a subject forcancer comprising administering to the subject decitabine; andadministering to the subject a pharmaceutical composition comprising acompound of any one of formulae I-VIII. The decitabine and the compoundgiven can be any one of formulae I-VIII and can be administered to thesubject sequentially or simultaneously. A sequential administrationincludes (a) first administering decitabine followed by (b)administering the pharmaceutical composition comprising a compound ofany one of formulae I-VIII. An alternative sequential administrationincludes (a) first administering the pharmaceutical compositioncomprising a compound of any one of formulae I-VIII followed by (b)administering decitabine. A simultaneous administration includesadministering decitabine and the pharmaceutical composition comprising acompound of any one of formulae I-VIII at the same time; or atsubstantially the same time.

When administration involves the separate administration (e.g.,sequential administration) of the first compound (e.g., a compound ofFormula I) and a second compound (e.g., decitabine), as describedherein, the compounds are administered sufficiently close in time tohave the desired therapeutic effect. For example, the period of timebetween each administration, which can result in the desired therapeuticeffect, can range from minutes to hours to days and can be determinedbased on the properties of each compound such as potency, solubility,bioavailability, plasma half-life and kinetic profile. For example, thecompounds can be administered in any order within 24-72 hours of eachother or within any time less than 24 hours of each other.Alternatively, the compounds can be administered in any order within oneweek of each other.

When decitabine and the compound of any one of formulae I-VIII areadministered sequentially, they are separately formulated and can beprovided in any order. When decitabine and the compound of any one offormulae I-VIII are administered simultaneously, however, they may beeither separately formulated or combined in the same formulation. Whencombined in the same formulation, decitabine and the compound of any oneof formulae I-VIII can be formulated so as to be released into thesubject at the same time or at different times. The release profile of aformulation comprising both decitabine and the compound of any one offormulae I-VIII includes the following:

-   -   A) release and bioavailability of decitabine followed by release        and bioavailability of the compound of any one of formulae        I-VIII;    -   B) release and bioavailability of the compound of any one of        formulae I-VIII followed by release and bioavailability of        decitabine;    -   C) release and bioavailability of the compound of any one of        formulae I-VIII at the same time as (or substantially at the        same time as) release and bioavailability of decitabine.

Thus, provided herein is a method of treating cancer, comprisingadministering to a subject in need thereof a composition comprisingdecitabine and a compound of any one of formulae I-VIII. The cancer tobe treated can be chronic myelocytic leukemia, melanoma, myelodysplasia,relapsed leukemia, colon cancer (including colorectal cancer),gastrointestinal cancer, ovarian cancer, acute lymphoid leukemia, acutemyeloid leukemia, lymphocytic leukemia, carcinoma of the prostate,chronic myeloid leukemia, colorectal cancer, non-small cell lung cancer,prostate tumor, renal cell carcinoma, testicular cancer, breast cancer,fallopian tube cancer, ovary tumor, peritoneal tumor, neuroblastoma,non-Hodgkin's lymphoma, head and neck tumor, small intestine cancer,esophagus tumor, lung tumor (or, lung cancer), small cell lung cancer,or mesothelioma. In a particular embodiment, the cancer to be treated ismyelodysplastic syndrome, acute myelogenous leukemia, or chronicmyelocytic leukemia.

In another embodiment, provided herein is a method of treating cancer ina subject in need thereof, comprising administering to the subject acomposition comprising decitabine and ER-876437. In still anotherembodiment, provided herein is a method of treating cancer in a subjectin need thereof, comprising administering to the subject a compositioncomprising decitabine and ER-876437, wherein the cancer is selected fromthe group consisting of myelodysplastic syndrome, leukemia, pancreaticcancer, ovarian cancer, peritoneal cancer, non small cell lung cancer,and metastatic breast cancer. In still another embodiment, providedherein is a method of treating acute myelogenous leukemia,myelodysplastic syndrome or chronic myelocytic leukemia in a subject inneed thereof, comprising administering to the subject a compositioncomprising decitabine and ER-876437.

In still another embodiment, provided herein is a method of treatingsickle cell anemia in a subject in need thereof, comprisingadministering to the subject a composition comprising decitabine andER-876437. In still another embodiment, provided herein is a method oftreating postallogeneic progenitor cell transplant relapse in a subjectin need thereof, comprising administering to the subject a compositioncomprising decitabine and ER-876437.

In another embodiment of the invention, the decitabine and the compoundof any one of formulae I-VIII can be administered sequentially (in anyorder) or simultaneously with other pharmaceutical agents typicallyadministered to subjects being treated for cancer. Such otherpharmaceutical agents include without limitation anti-emetics, agentsthat increase appetite, other cytotoxic or chemotherapeutic agents, andagents that relieve pain. The decitabine and the compound of any one offormulae I-VIII can be formulated together with or separately from suchother pharmaceutical agents.

A combination with such other pharmaceutical agents can either result insynergistic increase in anti-cancer activity, or such an increase can beadditive. Compositions described herein typically include lower dosagesof each compound in a composition, thereby avoiding adverse interactionsbetween compounds or harmful side effects, such as ones which have beenreported for similar compounds. Furthermore, normal amounts of eachcompound when given in combination could provide for greater efficacy insubjects who are either unresponsive or minimally responsive to eachcompound when used alone.

In certain embodiments, the invention provides a pharmaceuticalcomposition of any of the compositions of the present invention. In arelated embodiment, the invention provides a pharmaceutical compositionof any of the compositions of the present invention and apharmaceutically acceptable carrier or excipient of any of thesecompositions. In certain embodiments, the invention includes thecompositions as novel chemical entities.

In one embodiment, the invention includes a packaged cancer treatment.The packaged treatment includes a composition of the invention packagedwith instructions for using an effective amount of the composition ofthe invention for an intended use. In other embodiments, the presentinvention provides a use of any of the compositions of the invention formanufacture of a medicament to treat cancer infection in a subject.

Synthetic Procedure

Within the scope of this text, a readily removable group that is not aconstituent of the particular desired end product of the compounds ofthe present invention is designated a “protecting group.” The protectionof functional groups by such protecting groups, the protecting groupsthemselves, and their cleavage reactions are described for example instandard reference works, such as e.g., Science of Synthesis:Houben-Weyl Methods of Molecular Transformation. Georg Thieme Verlag,Stuttgart, Germany. 2005. 41627 pp. (URL:http://www.science-of-synthesis.com (Electronic Version, 48 Volumes));J. F. W. McOmie, “Protective Groups in Organic Chemistry”, Plenum Press,London and New York 1973, in T. W. Greene and P. G. M. Wuts, “ProtectiveGroups in Organic Synthesis”, Third edition, Wiley, New York 1999, in“The Peptides”; Volume 3 (editors: E. Gross and J. Meienhofer), AcademicPress, London and New York 1981, in “Methoden der organischen Chemie”(Methods of Organic Chemistry), Houben Weyl, 4th edition, Volume 15/I,Georg Thieme Verlag, Stuttgart 1974, in H.-D. Jakubke and H. Jeschkeit,“Aminosduren, Peptide, Proteine” (Amino acids, Peptides, Proteins),Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982, and in JochenLehmann, “Chemie der Kohlenhydrate: Monosaccharide and Derivate”(Chemistry of Carbohydrates: Monosaccharides and Derivatives), GeorgThieme Verlag, Stuttgart 1974. A character-istic of protecting groups isthat they may be removed readily (i.e., without the occurrence ofundesired secondary reactions) for example by solvolysis, reduction,photolysis or alternatively under physiological conditions (e.g., byenzymatic cleavage).

Acid addition salts of the compounds of the invention are most suitablyformed from pharmaceutically acceptable acids, and include for examplethose formed with inorganic acids, e.g., hydrochloric, hydrobromic,sulphuric or phosphoric acids and organic acids, e.g., succinic, maleic,acetic or fumaric acid. Other non-pharmaceutically acceptable salts,e.g., oxalates may be used for example in the isolation of the compoundsof the invention, for laboratory use, or for subsequent conversion to apharmaceutically acceptable acid addition salt. Also included within thescope of the invention are solvates and hydrates of the invention.

The conversion of a given compound salt to a desired compound salt isachieved by applying standard techniques, in which an aqueous solutionof the given salt is treated with a solution of base e.g. sodiumcarbonate or potassium hydroxide, to liberate the free base which isthen extracted into an appropriate solvent, such as ether. The free baseis then separated from the aqueous portion, dried, and treated with therequisite acid to give the desired salt.

In vivo hydrolyzable esters or amides of certain compounds of theinvention may be formed by treating those compounds having a freehydroxy or amino functionality with the acid chloride of the desiredester in the presence of a base in an inert solvent such as methylenechloride or chloroform. Suitable bases include triethylamine orpyridine. Conversely, compounds of the invention having a free carboxygroup may be esterified using standard conditions which can includeactivation followed by treatment with the desired alcohol in thepresence of a suitable base.

Mixtures of isomers obtainable according to the invention may beseparated in a manner known per se into the individual isomers;diastereoisomers may be separated, for example, by partitioning betweenpolyphasic solvent mixtures, recrystallisation or chromatographicseparation, for example over silica gel or by, e.g., medium pressureliquid chromatography over a reversed phase column, and racemates may beseparated, for example, by the formation of salts with optically puresalt-forming reagents and separation of the mixture of diastereoisomersso obtainable, for example by means of fractional crystallisation, or bychromatography over optically active column materials.

Intermediates and final products may be worked up or purified accordingto standard methods, e.g., using chromatographic methods, distributionmethods, (re-) crystallization, and the like.

Methods of preparing gemcitabine are known in the art. Methods ofpreparing decitabine are known in the art.

In another embodiment, the invention is directed to a method of couplingcyclic urea compounds such as imidazolidin-2-one,tetrahydropyrimidin-2(1H)-one, 1,3-diazepan-2-one or1,3,4,7-tetrahydro-2H-1,3-diazepin-2-one (ER-878899) to aC-2-substituted tetrahydrofuran ring comprising forming a reactionmixture by mixing (i) a first solution comprising the1,3,4,7-tetrahydro-2H-1,3-diazepin-2-one in a reaction solvent with (ii)a second solution comprising the C-2-substituted tetrahydrofuran ring inthe reaction solvent under reflux conditions. In this embodiment, thereflux conditions can maintain the volume of the reaction mixture as thefirst solution is added to the second solution. Alternatively, thereflux conditions can prevent the volume of the reaction mixture fromincreasing by more than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2% or 1%.In this embodiment, the reaction solvent can be a polar, aprotic solventhaving a boiling point greater than 150° C., such as dimethylacetamide(DMA) or dimethylsulfoxide (DMSO). According to this embodiment, thesecond solution is heated to greater than 150° C., and the firstsolution can be added via syringe to the second solution. According tothis embodiment, the first solution can be added to the second solutionover a time period extending less than 10 hours, less than 5 hours, lessthan 3 hours, less than 2 hours, less than 1 hour or less than 30minutes. According to this embodiment, the second solution can be heatedfrom 150° C. to 250° C., from 175° C. to 225° C., or from 200° C. to220° C. According to this embodiment, the C-2-substitutedtetrahydrofuran ring can have substituents in the C-3 position, whichcan include one halogen in the C-3 position, two halogens in the C-3position, or two fluorines in the C-3 position. According to thisembodiment, the tetrahydrofuran ring can be ER-878898. With theexception of mutually exclusive values, any of the alternative featuresdescribed in this paragraph can be used together.

In another embodiment, the invention is directed to a method ofisolating ER-879381 from a mixture comprising ER-878617 comprising (i)contacting the mixture with a chromatographic substance, and separatingthe mixture on the substance using toluene and acetonitrile as themobile phase. According to this embodiment, the chromatographicsubstance can be silica gel. According to this embodiment, the mobilephase can be toluene:acetonitrile in a 7:1 ratio. Alternatively,according to this embodiment, the toluene:acetonitrile can have a ratioof greater than 7:1, or less than 7:1. With the exception of mutuallyexclusive values, any of the alternative features described in thisparagraph can be used together.

Dosage Forms

In certain other embodiments, the compositions of the instant invention(e.g., a compound of formula I in combination with decitabine or acompound of formula I in combination with a non-decitabine CDAsubstrate, e.g., ER-876437 in combination with gemcitabine) may beadministered to a subject in need thereof using the formulations andmethods described in U.S. Pat. No. 6,001,994, U.S. Pat. No. 6,469,058,and U.S. Pat. No. 6,555,518, and all of which are incorporated herein byreference in their entireties.

In certain embodiments, the compositions of the instant invention (e.g.,a compound of formula I in combination with decitabine, e.g., ER-876437in combination with decitabine) can be administered to a subject in needthereof using the formulations and methods described in U.S. Pat. No.7,144,873, U.S. Pat. No. 7,135,464, U.S. Pat. No. 6,982,253, U.S. Pat.No. 6,905,669, and U.S. Pat. No. 6,613,753, all of which areincorporated herein by reference in their entireties.

In some embodiments, pharmaceutical compositions of the compounds (orcombinations) of the invention may be in unitary dosage form suitablefor administration orally, rectally or by parenteral injection. Forexample, in preparing compositions in oral dosage form, any of the usualpharmaceutical media may be employed, such as, for example, water,glycols, oils, alcohols and the like, as in the case of oral liquidpreparations such as suspensions, syrups, elixirs and solutions; orsolid carriers such as starches, sugars, kaolin, lubricants, binders,disintegrating agents and the like in the case of powders, pills,capsules and tablets. Because of their ease in administration, tabletsand capsules represent the most advantageous oral dosage unit form, inwhich case solid pharmaceutical carriers are employed. For parenteralcompositions, carriers usually comprise sterile water, at least in largepart, though other ingredients, for example, to aid solubility, may beincluded. Injectable solutions, for example, are prepared using acarrier which comprises saline solution, glucose solution or a mixtureof saline and glucose solution. Injectable suspensions may also beprepared in which case appropriate liquid carriers, suspending agentsand the like may be employed. In case of compositions suitable forpercutaneous administration, carrier optionally comprises a penetrationenhancing agent or a suitable wetting agent, which may be combined withsuitable additives of any nature in minor proportions, which additivesdo not cause a significant deleterious effect to the skin. Additives mayfacilitate the administration to the skin or may be helpful forpreparing desired compositions. These compositions may be administeredin various ways, e.g., as a transdermal patch, as a spot-on, as anointment.

It is especially advantageous to formulate the pharmaceuticalcompositions described herein in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form, as usedherein, refers to physically discrete units suitable as unitary dosages,each unit containing a predetermined quantity of active ingredientcalculated to produce the desired therapeutic effect in association withthe required pharmaceutical carrier. Examples of such dosage unit formsare tablets (including scored or coated tablets), capsules, pills,powder packets, wafers, injectable solutions or suspensions,teaspoonfuls, tablespoonfuls and the like, and segregated multiplesthereof.

In general it is contemplated that a therapeutically effective amount ofa first or a second compound would be from 0.0001 mg/kg to 0.001 mg/kg;0.001 mg/kg to 10 mg/kg body weight or from 0.02 mg/kg to 5 mg/kg bodyweight. In some embodiments, a therapeutically effective amount of afirst or a second compound is from 0.007 mg to 0.07 mg, 0.07 mg to 700mg, or from 1.4 mg to 350 mg. A method of prophylactic or curativetreatment may also include administering the composition in a regimen ofbetween one to five intakes per day.

In some embodiments, a therapeutically effective amount of a firstcompound or a second compound includes, but is not limited to, theamount less than 0.01 mg/dose, or less than 0.5 mg/dose, or less than 1mg/dose, or less than 2 mg/dose, or less than 5 mg/dose, or less than 10mg/dose, or less than 20 mg/dose, or less than 25 mg/dose, or less than50 mg/dose, or less than 100 mg/dose, or less than 500 mg/dose. Thenumber of times a day a first or a second compound is administrated to asubject may be determined based on various criteria commonly used in theart or those described herein.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, α-tocopherol, and the like; and metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical, buccal, sublingual, rectal, vaginal or parenteraladministration. The formulations may conveniently be presented in unitdosage form and may be prepared by any methods well known in the art ofpharmacy. The amount of active ingredient that may be combined with acarrier material to produce a single dosage form will generally be thatamount of the composition that produces a therapeutic effect. Generally,out of one hundred percent, this amount will range from about 1 percentto about ninety-nine percent of active ingredient, preferably from about5 percent to about 70 percent, most preferably from about 10 percent toabout 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association a composition of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a composition of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) or as mouth washes and the like,each containing a predetermined amount of a composition of the presentinvention as an active ingredient. A composition of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, or any of thefollowing: fillers or extenders, such as starches, lactose, sucrose,glucose, mannitol, or silicic acid; binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose or acacia; humectants, such as glycerol; disintegrating agents,such as agar-agar, calcium carbonate, potato or tapioca starch, alginicacid, certain silicates, and sodium carbonate; solution retardingagents, such as paraffin; absorption accelerators, such as quaternaryammonium compounds; wetting agents, such as, for example, cetyl alcoholand glycerol monostearate; absorbents, such as kaolin and bentoniteclay; lubricants, such a talc, calcium stearate, magnesium stearate,solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof;and coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compositionmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions that may bedissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions that may be used include polymeric substances andwaxes. The active ingredient can also be in micro-encapsulated form, ifappropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compositions of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluent commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compositions, may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compositions of theinvention with one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active composition.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of acomposition of this invention include powders, sprays, ointments,pastes, creams, lotions, gels, solutions, patches and inhalants. Theactive composition may be mixed under sterile conditions with apharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants that may be required.

The ointments, pastes, creams and gels may contain, in addition to anactive composition of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a composition of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a composition of the present invention to the body. Suchdosage forms may be made by dissolving or dispersing the composition inthe proper medium. Absorption enhancers can also be used to increase theflux of the composition across the skin. The rate of such flux may becontrolled by either providing a rate controlling membrane or dispersingthe active composition in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compositions of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity may be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents that delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compositions in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease may be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissue.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given by formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc., administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Oral or IV administration is preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, or thepharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methods.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds or materials used in combination with the particularcompound employed, the age, sex, weight, condition, general health andprior medical history of the patient being treated, and like factorswell known in the medical arts.

A physician or veterinarian can determine and prescribe the effectiveamount of the pharmaceutical composition required. For example, thephysician or veterinarian could start doses of the compounds of theinvention employed in the pharmaceutical composition at levels lowerthan that required in order to achieve the desired therapeutic effectand gradually increase the dosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound that is the lowest dose effective to producea therapeutic effect. Such an effective dose will generally depend uponthe factors described above. Generally, intravenous and subcutaneousdoses of the compounds of this invention for a patient, when used forthe indicated analgesic effects, will range from about 0.0001 to about100 mg per kilogram of body weight per day, more preferably from about0.01 to about 50 mg per kg per day, and still more preferably from about1.0 to about 100 mg per kg per day. An effective amount is that amounttreats a viral infection.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical composition.

EXAMPLES

General methods and experimentals for preparing compounds of the presentinvention are set forth below.

Example I Chemical Syntheses

Unless otherwise stated, for Examples I.B.-I.C., solvent removal wascarried out using a Mai rotary evaporator. Analytical chromatography wascarried out using a Hewlett Packard series 1100 HPLC and preparativechromatography was carried out using either Biotage SP4 instrument or aWaters 4000 instrument using Chiralpak IA columns under neutralcondition, unless indicated otherwise. Mass spectra were recorded usingWaters Acquity UPLC/MS system. Like or comparable equipment was used forthe remaining examples.

NMR spectra were recorded using a Varian 400 MHz spectrometer (ExamplesI.B.-I.C.) or using a Fluka 400 MHz spectrometer (Examples I.A. andI.D.).

Example I.A. ER-876437 I.A.1: Preparation of ER-878899(1,3,4,7-tetrahydro-2H-1,3-diazepin-2-one)

ER-878899 was prepared as outlined in Scheme I below. This preparationwas described in J. Med. Chem. 1981, 24, 662-666; J. Org. Chem. 1980,45, 485-489 and Bull. Soc. Chim. Fr. 1973, 198-292, all of which arehereby incorporated by reference in their entirety.

Mechanical stirring is required for the formation of ER-878899 madeaccording to Scheme I. Carbonyl sulfide may be bubbled into the reactionflask using a glass pipette (of large diameter) and not a needle, whichtends to clog due to the solid formed during the reaction. At the end ofthe reaction, the insoluble material in the reaction medium wasfiltered, and ER-878899 may be present in the filter cake.

I.A.2.: Preparation of ER-876437

ER-878899, prepared according to I.A.1, was used in Scheme II asdescribed below.

1-(3,3-Difluoro-4-benzoyl-5-benzoxymethyl-tetrahydro-furan-2-yl)-1,3,4,7-tetrahydro-[1,3]diazepin-2-one(ER-879381)

The commercially available mesylate ER-878898 shown above in Scheme II(3.8 g, 8.3 mmol) and the urea ER-878899 (900 mg, 8.0 mmol) were addedto dimethylacetamide (DMA) (400 ml). Upon heating (170° C.), thereaction components solubilized. The solution was heated overnight (15h) under an atmosphere of nitrogen.

The DMA was then removed in vacuo. The residue was resuspended in EtOAc(150 ml) and then washed with water (2×75 ml). The combined organiclayers were dried over MgSO₄, filtered, and concentrated in vacuo. Thematerial was chromatographed on SiO₂ and was eluted with 50%EtOAc/hexanes. The material obtained after chromatography was theunresolved α/β anomers. The anomers were then separated using normalphase preparative HPLC (50% EtOAc/hexanes isocratic, 10 ml/min, Rt=25.7min.); column: phenomenex luna 10μ Silica 100A, 250×21.20 mm; refractiveindex detector. The β anomer ER-879381 was isolated in >90% purity (10%a anomer, Rt. 24 min). ¹H NMR (CDCl₃) δ 8.05 (m, 4H), 7.59 (m, 2H), 7.43(m, 4H), 5.99 (m, 1H), 5.72 (m, 2H), 5.54 (m, 1H), 4.77 (dd, J=12.1, 3.4Hz, 1H), 4.65 (br s, 1H), 4.56 (dd, J=12.4, 4.0 Hz, 1H), 4.38 (m, 1H),3.80 (m, 4H).

1-(3,3-Difluoro-4-hydroxy-5-hydroxymethyl-tetrahydro-furan-2-yl)-1,3,4,7-tetrahydro-[1,3]diazepin-2-one(ER-876437)

ER-879381 was dissolved in NH₃ (7M) in MeOH (40 ml). The solutionstirred overnight. The solvent was removed and the residue was purifiedby RP HPLC (10% acetonitrile/H₂O, flow 10 ml/min, Rt=23 minutes);column: phenomenex luna 5μ C18(2) 100A, 250×21.2 mm; refractive indexdetector. The desired compound ER-876437 was obtained in 1.5% (62 mg)overall yield. ¹H NMR (D₂O) δ 5.86 (m, 2H), 5.69 (dd, J=14.3 Hz, 6.2 Hz,1H), 4.14 (m, 1H), 3.86 (m 1H), 3.74 (m, 6H). ¹³C NMR (D₂O) δ 164.5,127.3, 126.2, 122.1 (dd, J=252, 261 Hz, 1C), 85.9 (dd, J=41, 22 Hz, 1C),77.4 (d, J=8 Hz, 1C), 69.5 (dd, J=22 Hz, 19 Hz, 1C), 58.9, 41.0, 40.7.

The carbon, hydrogen and nitrogen components of the molecular formula(C₁₀H₁₄N₂O₄F₂+0.5H₂O) was calculated to be C, 43.96; H, 5.53; and N,10.25. Elemental analysis revealed this material to contain C, 43.99; H,5.36; and N, 10.21.

Marginal improvements to the yield of the coupling reaction of ER-878899to the mesylate may be obtained by changing the reaction solvent. Whendiglyme is used as the solvent, a 15% yield improvement may be observed.

Example I.B. ER-876437 I.B.1.: Preparation of ER-878899(1,3,4,7-tetrahydro-2H-1,3-diazepin-2-one)

ER-878705 (shown below) was prepared following the procedure describedin Feigenbaum, A. and Lehn, J. M., Bull. Soc. Chim. Fr., 1973, 198-202and Liu, P. S., Marquez, V. E., Driscoll, J. S, and Fuller, R. W., J.Med. Chem., 1981, 24, 662-666.

To a white suspension of ER-878705 (79.7 g, 230 mmol) in ethanol (470mL) in a two-neck 2 L flask equipped with mechanical stirrer was addedhydrazine hydrate (23.5 mL, 483 mmol) at room temperature. The resultingwhite suspension was heated to 50° C. for 30 minutes to obtain a clearlight yellow solution. As white precipitate started appearing, themixture was heated to 60° C. for 3 hours and the stirring became verydifficult. After allowing the mixture to cool to room temperature,concentrated hydrogen chloride solution (40.3 mL, 483 mmol) was addedand the mixture became easily stirred. After stirring for 30 minutes,the mixture was filtered and washed with 5×200 mL of water. The filtratewas concentrated to a dry solid. The dry solid was suspended in 200 mLof ethanol, and stirred for 1 hour to make a nice suspension. Thesuspension was filtered and washed with 3×100 mL pure ethanol. The cake(white granular-like crystal) was collected and dried to give 34.6 (94%)g of 1,4-diamino-2-butene di-hydrochloride salt. ¹H NMR showed theproduct contains phthalhydrazide as a minor impurity in the ratio of5:1. ¹H NMR (400 MHz, CD₃OD) δ 5.85 (ddd, J=1.6, 1.8 and 4.4 Hz, 2H),3.69 (d, J=4.4, 4H).

To a suspension of 1,4-diamino-2-butene di-hydrochloride salt (22.7 g,143 mmol) in ethanol (1.2 L) in a two-neck 2 L flask was added 1.0 MNaOH solution (330 mL, 330 mmol). Upon addition of NaOH to thesuspension, the mixture became a transparent and colorless solution. Thesolution was heated to 70° C. and carbonyl sulfide was bubbled throughthe heated mixture. Thereafter, the mixture was heated to 80° C. atreflux. After 3 hours, the bubbling was stopped and the mixture washeated an additional 1.5 hours, cooled to room temperature andneutralized by addition of 1.0 N HCl (50 mmol). The mixture wasconcentrated to a dry gray solid. The solid was suspended in 1 L ofmethanol, stirred for 2 hours, filtered and washed with methanol. Thefiltrate was concentrated to about 200 mL volume, cooled to 0° C.,filtered and washed with cold methanol. The solid was collected anddried to give 5.05 g product. ¹H NMR showed it contained very minorimpurity phthalhydazide in the ratio of 13:1. ¹H NMR (400 MHz, CD3OD) δ5.91 (ddd, J=0.8, 1.2 and 1.6 Hz, 2H), 3.67 (d, J=4.0, 4H). The motherliquor was concentrated to about 30 mL, cooled to −10° C., filtered andwashed with cooled MeOH (−10° C.). The solid was collected and dried togive 7.10 g of product with minor contamination of phthalhydrazide inthe ratio of 4:1 as determined by ¹H NMR.

I.B.2.: Preparation of ER-878617

As depicted in Scheme V above, a solution of ER-878898 (1.33 g, 2.92mmol, available from Waterstone or Depew Fine Chemical) and ER-878899(200.0 mg, 1.78 mmol) in dry DMA (30 mL) was heated and stirred at180-190° C. (oil bath temperature) as DMA distilled out slowly.Additional azeotroped 1,3,4,7-tetrahydro-2H-1,3-diazepin-2-one (800.0mg, 7.13 mmol) in DMA (50 mL) was added with syringe pump over 2 hoursduring this DMA distillation. After addition of all material, thereaction was kept at reflux for 30 minutes and allowed to cool down. Thereaction mixture was concentrated in vacuo and the residue was purifiedwith chromatography to give ER-878617 (624.8 mg, 45%) as a mixture oftwo epimers.

I.B.3.: Preparation of ER-876437

As depicted in Scheme VI above, a solution of ER-878617 (624.8 mg, 1.32mmol) in 7 M ammonia/methanol (53 mL) was stirred at ambient temperaturefor 18 hours. The reaction mixture was concentrated in vacuo and theresidue was purified with preparative TLC to give a crude product (274.2mg, 78%) as the mixture of two epimers. The mixture of two epimers wereseparated on preparative chromatography with Chiralpak IA column (DaicelChemical Industries, Ltd., Tokyo Japan) to give ER-876437 (160.2 mg).

Example I.C. ER-876437 I.C.1.: Preparation of ER-879381

ER-879381 was made according to Scheme VII as shown below. ER-878899 wasprepared as described above in Example I.B.1.

As depicted in Scheme VII above, a solution of ER-878898 (8.0 g, 18mmol, available from Waterstone or Depew Fine Chemical) and ER-878899(1.2 g, 10.7 mmol) in dry DMA (100 mL) was heated and stirred at200-220° C. (oil bath temperature) as DMA distilled out slowly.Additional azeotroped 1,3,4,7-tetrahydro-2H-1,3-diazepin-2-one (4.8 g,42.9 mmol) in DMA (350 mL) was added through syringe pump over 2 hoursduring this DMA distillation. After addition of all material, thereaction was kept at reflux for 30 minutes and allowed to cool down. Thereaction mixture was concentrated in vacuo and the residue was combinedwith the residue from a separate experiment conducted on the same scaleusing the same procedure. The combined residue was purified with silicagel chromatography (mobile phase: 50-100% AcOEt/Heptane) to give amixture of two epimers (9.38 g). The mixture of two epimers was furtherseparated with silica gel chromatography (mobile phase:toluene:acetonitrile=7:1) to produce ER-879381 (3.94 g).

I.C.2.: Preparation of ER-876437

ER-876437 was prepared as shown below in Scheme VIII.

As depicted in Scheme VIII above, a solution of ER-879381 (3.8 g, 8.0mmol) in 7 M ammonia/methanol (100 mL) was stirred at ambienttemperature for 17 hours. The reaction mixture was concentrated in vacuoand the residue was purified with chromatography (mobile phase: 50-100%AcOEt/Heptane) to give ER-876437 (1.89 g, yield 89%).

Example I.D. ER-878895 I.D.1.: Preparation of ER-878890

As depicted in Scheme IX above, a solution of ER-878889 (preparedaccording to Stimac, A. and Kobe, J., Carbohydr. Res., 2000, 329,317-324, 4.3 g, 11.7 mmol) and di-tert-butyldicarbonate (5.4 g, 24.6mmol) in THF (125 mL) was stirred in the presence of Lindlar's catalyst(1 g) at 30 psi over the weekend. The reaction suspension containing thehydrogenated product was filtered through Celite and concentrated. Theresidue was purified with radial chromatography to give ER-878890 (2.8g). ER-878890 was further purified by recrystallization fromAcOEt/Hexane to give white needles with mp 106-108° C.

I.D.2.: Preparation of ER-878891

As depicted in Scheme X above, to a stirring solution of ER-878890 (1.6g, 3.48 mmol) in THF/DMF (100 mL/30 mL) was added 0.5 M potassiumhexamethydisilazide (KHMDS) in toluene (8.5 mL, 4.25 mmol) dropwise atabout −78° C. (dry ice/acetone bath), followed by addition of allylbromide (0.4 mL, 4.6 mmol). The reaction mixture was stirred overnightas the dry ice-acetone bath slowly warmed to room temperature (˜25° C.).The reaction was quenched with saturated aqueous ammonium chloride andextracted with AcOEt. The organic phase was washed with brine and driedover anhydrous magnesium sulfate. The dried solution was filtered andevaporated. The residue was purified with radial chromatography to giveER-878891 (0.64 g).

I.D.3.: Preparation of ER-878892

As depicted in Scheme XI above, to a stirring solution of ER-878891 (0.1g, 0.2 mmol) in dichloromethane (DCM) (1 mL) under nitrogen was addedtrifluoroacetic acid (TFA) (0.5 mL) at room temperature. ER-878891disappeared in 1 hour and the solvent and TFA were evaporated in vacuo.To the resulting oil redissolved in DCM (2 mL) was added allylisocyanate (0.2 mL, 2.2 mmol) at room temperature. The reaction mixturewas evaporated after 1 hour and purified by radial chromatography togive ER-878892 (50% yield) as a mixture of two anomers (beta/alpha˜3/1).

I.D.4.: Preparation of ER-878893

As depicted in Scheme XII above, to a stirring solution of ER-878892(0.27 g, 0.56 mmol) in THF (10 mL) under nitrogen was added 0.5M KHMDSin toluene (1.5 mL, 0.75 mmol) at about −78° C. (dry ice/acetone bath),followed by addition of benzoyl chloride (0.6 mL, 5.1 mmol). Thereaction mixture was stirred overnight and allowed to slowly warm toroom temperature. The reaction was quenched with saturated aqueousammonium chloride and extracted with AcOEt. The organic phase was washedwith brine, dried over anhydrous magnesium sulfate, filtered andevaporated. The residue was purified with radial chromatography to giveER-878893 (0.13 g, 50% yield) as a mixture of anomers.

I.D.5.: Preparation of ER-878894

As depicted in Scheme XIII above, to a degassed solution of ER-878893(0.13 g, 0.22 mmol) in DCM (120 mL) was added Grubb's 2^(nd) generationcatalyst (˜30 mg, available from Sigma-Aldrich, St. Louis, Mo.) undernitrogen. This catalyst affords the ring closing metathesis (RCM). Thereaction mixture was heated at 40° C. for 1 hour followed by evaporationof the solvent. To the residue dissolved in AcOEt (20 mL) was addedSilicycle Si-triamine Pd scavenger (Silicycle Inc.) and stirredvigorously for 1 hour. The reaction mixture was filtered andconcentrated. The resulting pale yellow viscous oil was purified withradial chromatography and the less polar compound was determined to beER-878894 (40 mg) which crystallized on standing.

I.D.6.: Preparation of ER-878895

As depicted in Scheme XIV above, a solution of ER-878894 (65 mg, 0.14mmol) in 0.1N NaOH/MeOH (3 mL) was stirred for 30 minutes until all ofthe UV active spots disappeared by TLC. The solvent was removed in vacuoand the crude solid was dissolved in water (2 mL). The solution wasneutralized with HCl and the solvent was removed in vacuo. The residuewas purified by reverse phase preparative HPLC to afford ER-878895 (12mg, 35%).

Table 1 provides analytical data for compounds described herein.

TABLE 1 Analytical Data Structure ER-# Analytical Data

878617 Salt free ¹H NMR: (400 MHz, CDCl₃) δ 8.05 (m, 4H), 7.55 (m, 2H),7.45 (m, 4H), 6.22 (t, J = 10.4 Hz), 5.95 (dd, J = 12.8, 10.6 Hz),5.88-5.66 (m), 5.5 (m), 4.76 (dd, J = 12.4, 3.6 Hz), 4.65 (m), 4.55 (m),4.55 (dd, J = 12, 4.4 Hz), 4.36 (m), 3.94-3.64 (m) MS (ESI) m/z 473.31(M + H)⁺

879381 Salt free ¹H NMR: (400 MHz, CDCl₃) δ 8.05 (m, 4H), 7.64 (m, 2H),7.48 (m, 4H), 6.04 (dd, J = 12.0, 10.4 Hz, 1H), 5.76 (m, 2H), 5.58 (ddd,J = 12.0, 6.4, 5.2 Hz, 1H), 4.81 (dd, J = 12.4, 3.6 Hz, 1H), 4.61 (dd, J= 12.8, 4.4 Hz, 1H), 4.58 (broad, partially overlap with 4.61 peaks,1H), 4.43 (dt, J = 6.4, 3.4 Hz, 1H), 3.99-3.71 (m, 4H)

876437 Salt free ¹H NMR: (400 MHz, CD₃OD) δ 5.83 (m, 2H), 5.69 (dd, J =21.2, 8.0 Hz, 1H), 4.05 (ddd, J = 14.0, 11.2, 8.4 Hz, 1H), 3.86-3.58 (m,7H) MS (ESI) m/z 265.17 (M + H)⁺

878890 Salt free ¹H NMR: (400 MHz, CDCl₃) δ 8.11-8.00 (m, 4H), 7.66-7.53(m, 2H), 7.52-7.40 (m, 4H), 5.86 (dd, J = 16, 10 Hz, 1H), 5.57 (d, J =18 Hz, 1H), 5.41 (s, 1H), 5.30 (s, 1H), 5.23 (d, J = 50 Hz, 1H), 4.60(s, 2H), 1.47 (s, 9H)

878891 Salt free ¹H NMR: (400 MHz, CDCl₃) δ 8.08 (d, J = 7.6 Hz, 2H),8.05 (d, J = 8.0 Hz, 2H), 7.62 (t, J = 7.6 Hz, 1H), 7.56 (t, J = 7.6 Hz,1H), 7.46 (m, 4H), 6.0 (dd, J = 18.4, 4.4 Hz, 1H), 5.88 (m, 1H), 5.71(dt, J = 19.6, 3.2 Hz, 1H), 5.48 (d, J = 52 Hz, 1H), 5.18 (d, J = 17.2Hz, 1H), 5.14 (d, J = 10.8 Hz, 1H), 4.61 (broad s, 3H), 3.93 (m, 2H),1.47 (s, 9H)

878892 Salt free ¹H NMR: (400 MHz, CDCl₃) δ 8.11- 8.00 (m, 4H),7.66-7.53 (m, 2H), 7.52- 7.40 (m, 4H), 6.35 (dd, J = 26, 3 Hz, 1H), 6.06(dd, J = 18, 5 Hz, 1H), 6.00-5.79 (m, 3H), 5.67 (dt, J = 19, 4 Hz, 1H),5.58-5.50 (m, 1H), 5.44-4.95 (m, 8H), 4.74-4.53 (m, 4H), 4.01-3.97 (m,2H), 3.91-3.83 (m, 3H), 1.71 (s, 1H)

878894 Salt free ¹H NMR: (400 MHz, CDCl₃) δ 8.12 (dd, J = 8.2, 1.2 Hz,2H), 7.98 (dd, J = 8.2, 1.2 Hz, 2H), 7.6 (m, 5H), 7.45, (m, 6H), 5.93(dd, J = 24.4, 3 Hz, 1H), 5.79 (s, 2H), 5.59 (dd, J = 18.6, 3.2 Hz, 1H),5.14 (dd, J = 50.8, 2.8 Hz, 1H), 4.85 (d, J = 18.8 Hz, 1H), 4.81 (dd, J= 12, 3.8 Hz, 1H), 4.72 (dd. J = 12, 4.8 Hz, 1H), 4.35 (m, 2H), 4.17 (m,2H) MS (ESI) m/z 559.2 (M + H)⁺

878895 Salt free ¹H NMR: (400 MHz, D₂O) δ 5.8 (m, 2H), 5.7 (dd, J =18.4, 5.2 Hz, 1H), 4.93 (ddd, J = 53, 5.2, 3.8 Hz, 1H), 4.19 (ddd, J =22.8, 6.4, 3.6 Hz, 1H), 3.84-3.61 (m, 7H) MS (ESI) m/z 247.11 (M + H)⁺

Example II Assay for Inhibition of Cytidine Deaminase (CDA)

The cytidine deaminase (CDA) enzymatic assay described by Cacciamani, T.et al., Arch. Biochem. Biophys. 1991, 290, 285-92; Cohen R. et al., J.Biol. Chem., 1971, 246, 7566-8; and Vincenzetti S. et al., Protein Expr.Purif 1996, 8, 247-53 was used to determine the inhibitory activity(IC₅₀) of compounds described herein. Using this assay, the IC₅₀ ofthese compounds was determined by following the decrease of substrate(cytidine) caused by the deamination reaction catalyzed by CDA.Disappearance of substrate (cytidine) over time was monitored by theabsorbance at 280 nm of the reaction.

The assay reaction was carried out in potassium phosphate buffer (pH7.4, 20 mM, containing 1 mM DTT) in a total volume of 100 μl in a96-well plate format. The final reaction mixture contained cytidine (50μM) and purified human recombinant CDA. Purified enzyme was diluted soas to produce an absorbance change of approximately 2 milli-absorbanceunits/minute. Base line measurements of absorbance change over time weremade before substrate (cytidine) addition. After substrate addition,absorbance change was read every minute for 30 minutes with aFlexStation® 3 (Molecular Devices, Sunnyvale, Calif.). For eachcompound, 8 different concentrations (10 μM, 3.33 μM, 1.11 μM, 0.37 μM,0.12 μM, 0.041 μM, and 0.014 μM, and 0.0047 μM) were used to inhibit thereaction. The slopes of the absorbance change over time in each reactionwere calculated and used by the SoftMax® Pro 5 software (MolecularDevices, Sunnyvale, Calif.) to obtain IC₅₀ values.

TABLE 2 Inhibitory Potency of Test Compounds Structure ER-Number IC₅₀(nM)

876437 237 ± 86  n = 4

876400 101 ± 53  n = 4

878519 1616 ± 643  n = 3

878895 140 n = 1

876404 113 n = 2

Example III Pharmacokinetics of ER-876437 and ER-876400 in Mice After IVand PO Administrations

ER-876437 and ER-876400 were both administered to mice at 10 mg/kgintravenously (IV) via the tail vein, and at 10 mg/kg per os (PO, or,orally) via gastric gavage. All doses were prepared in phosphatebuffered saline (PBS) and were administered at a volume of 5 mL/kg. Fivemice per group were used in these studies. Blood samples were takenserially from the tail vein of each mouse at predetermined timepoints.Blood samples from all mice in each group were pooled together prior toprocessing for plasma. The pooled blood samples were spun down within30-60 minutes after withdrawal and the plasma was harvested and frozenfor assay. After preparation and extraction the samples were assayed byLC/MS/MS. The observed concentrations (ng/mL), are reported in Table 3below.

TABLE 3 Plasma concentrations (ng/mL) of ER-876437 and ER- 876400 inmice after IV and PO administrations ER-876437 ER-876400 Time (hr) IV POIV PO 0.167 11838 8597 19860 7101 0.5 7686 3720 10166 7859 1 3469 4179 4206 4665 2 1450 1145   1753 ^(a) 1750 4 214 146    495 ^(a) 320 6 18436  118 87 8 64 103   59 44 24 20 39   93 264 ^(a) Above thequantitation limit

The pharmacokinetic (PK) parameters of ER-876437 and ER-876400 werecalculated via non-compartmental analysis using Watson® v. 7.2. Theresulting PK parameters are presented in Tables 4 and 5 below:

TABLE 4 PK parameters of ER-876437 and ER-876400 in mice after IVadministrations Parameter Units ER-876437 ER-876400 Dose mg/kg 10.0 10.0t_(1/2) hr 6.1 16.1 AUC_(0-t) ng · hr/mL 12893 18838 AUC_(0-∞) ng ·hr/mL 13071 20999 AUC_(0-∞)/D ng · hr/mL/D 1307 2100 AUC_(Extrap) % 1.410.3 CL L/kg/hr 0.77 0.48 Vss L/kg 1.64 3.2

TABLE 5 PK parameters of ER-876437 and ER-876400 in mice after POadministrations Parameter Units ER-876437 ER-876400 Dose mg/kg    10.0   10.0 C_(max) ng/mL 8597 7859 t_(max) hr     0.167    0.5 AUC_(0-t) ng· hr/mL 8579 13160  AUC_(0-∞) ng · hr/mL 9499 NC AUC_(0-∞)/D ng ·hr/mL/D  950 NC AUC_(Extrap) %    9.7 NC t_(1/2) hr    16.3 NC F %   66.5 ^(a)    69.9 ^(a) ^(a) Calculated based on AUC_(0-t) NC = Notcalculated due to insufficient data

The results of the present study suggest that the PK profiles ofER-876437 and ER-876400 in male BALB-c mice are similar. Following 10mg/kg IV the PK of both ER-876437 and ER-876400 may be characterized bymoderate distribution (Vss=1.64 and 3.20 L/kg, respectively), slowclearance (CL=0.77 and 0.48 L/hr/kg, respectively), and slow elimination(t_(1/2)=6.1 and 16.1 hr, respectively).

The overall exposures (AUC_(0-∞)) after IV administration of ER-876437and ER-876400 to mice were 13071 and 20999 ng·hr/mL, respectively, whichresulted in dose-normalized exposures (AUC_(0-∞)/D) of respectively 1307and 2100 mL/g. Following 10 mg/kg PO, the C_(max) of ER-876437 andER-876400 were respectively 8597 and 7859 ng/mL, and were observed at at_(max) of respectively 1.0 and 2.0 hr. The AUC_(0-t) after POadministration of 10 mg/kg were 8579 and 13160 ng·hr/mL for ER-876437and ER-876400, respectively. The AUC_(0-∞) of ER-876437 was 9499ng·hr/mL and the t_(1/2) was 16.3 hr. Due to insufficient data in theterminal elimination phase these parameters could not be determined forER-876400. In addition, the t_(1/2) for ER-876437 after POadministration is roughly 2.5-fold higher that that after IVadministration.

The bioavailabilities (F %) of ER-876437 and ER-876400 were similar:66.5 and 69.9%, respectively.

In conclusion, the PK profiles of ER-876437 and ER-876400 in male BALB-cmice after a single IV or PO dose of 10 mg/kg are similar. It is noted,however, that under normal feeding conditions, mice have a high gastricpH of around 5. See Simpson, R. J et al. “Forms of soluble iron in mousestomach and duodenal lumen: significance for mucosal update,” BritishJournal of Nutrition. 63:79-89 (1990), which is hereby whollyincorporated by reference.

Example IV ER-876400 and ER-876437 Stability in Simulated Gastric Fluidat 37° C.

This example describes the stabilities of ER-876400 and ER-876437 insimulated gastric fluid having a pH of 1.45 at room temperature (˜25°C.) and at 37° C. For humans, under fasted conditions, the gastric pHhas been reported to range from 1.4 to 2.1. See Kararli, T. T.Comparison of the GI anatomy, physiology, and biochemistry of humans andcommonly used laboratory animals. BioPharm & DrugDispos. 16:351-380,1995, which is hereby wholly incorporated by reference. The gastric pHin fasted monkeys has been reported to have a similar range of 1-3. SeeKondo, H. et al. Characteristics of the gastric pH profiles of unfed andfed cynomolgus monkeys as pharmaceutical product development subjects.BioPharm & DrugDispos. 24:45-51, 2003, which is hereby whollyincorporated by reference.

Materials:

Simulated gastric fluid (SGF) was prepared by mixing the following into100 mL of HPLC grade (or purified) water: 200 mg of sodium chloride and1.87 mL of a 37.52% HCl stock solution.

Sample Preparation:

The initial (t=0) samples were prepared by respectively dilutingER-876400 or ER-876437 in water. All other samples were prepared bydissolving ˜2 mg of analyte (either ER-876400 or ER-876437) in ˜1.0 mLof simulated gastric fluid at 37° C.

The HPLC analyses were conducted using a Waters UPLC solvent deliverysystem with Corona CAD detection. The HPLC Column (Waters Atlantis HHST3 2.1×100 mm, 1.8 um) was maintained at 40° C. and preequilibrated witha solution containing 98% water and 2% acetonitrile. The temperaturecontrolled auto-sampler was maintained at 37° C. The flow rate for theWater/MeCN mobile phase was 0.65 mL/min, with a gradient followingsample injection (5 μL) as follows:

Gradient: Time (min) % Water % MeCN 0-2 98 2   2-2.5 linear gradientfrom (98% Water/2% MeCN) to (60% Water/40% MeCN) 2.5-3.5 60 40

Hence, in these HPLC-SGF degradation studies, a 5 μL aliquot was takenfrom the SGF/analyte solution at various times and loaded onto the HPLCcolumn with the above described features and conditions. The Water/MeCNmobile phase was applied to the column with the above described flowrate and gradient and the HPLC chromatograms were collected. After 3.5minutes, the column was reequilibrated with 98% water/2% MeCN for 1.5minutes.

HPLC chromatograms of either ER-876400 or ER-876437 in water affordedidentification of the peak attributed to ER-876400 or ER-876437. HPLCtraces of SGF without any ER-876400 or ER-876437 provided blank (orbackground) chromatograms that could be used to identify SGF-relatedpeaks and to distinguish those peaks from the analytes' peaks.Chromatograms were collected at the times identified in Tables 6 and 7,and the corresponding percentage of sample respectively attributed toeither ER-876400 or ER-876437 are provide for each sampling time. Theseresults are also depicted as plots in FIGS. 1 and 2.

TABLE 6 Stability of ER-876400 in SGF at 37° C. % ER-876400 AnalysisTime (peak retention (hours:minutes:seconds) time: 1.46 min) 0:00:0084.04 0:00:30 19.59 0:06:08 17.04 0:11:45 16.72 0:17:23 15.17 0:23:0114.20 0:28:38 13.23 0:34:16 12.51 0:39:54 14.05 0:45:33 11.42 0:51:1010.71 0:56:48 8.87 1:02:27 9.14 1:08:07 8.81 1:13:46 7.66 1:19:23 4.051:25:01 6.44 1:30:38 5.92 1:36:16 5.72 1:41:53 5.69 1:47:32 4.98 1:53:104.51 1:58:49 3.85 2:04:28 3.59 2:21:24 2.82 2:49:37 1.52 3:17:48 0.813:23:28 0.62 3:46:00 0.39 3:51:38 0.25

TABLE 7 Stability of ER-876437 in SGF at 37° C. % ER-876437 AnalysisTime (peak retention (Hours:Minutes:Seconds) time: 2.90 min) 0:00:0092.18 0:00:30 85.88 0:06:08 85.72 0:11:45 86.46 0:17:24 86.38 0:23:0283.22 0:28:39 83.48 0:34:17 83.80 0:39:54 84.16 0:45:32 82.62 0:51:1082.41 0:56:47 82.45 1:02:26 82.45 1:08:04 82.55 1:13:41 83.11 1:19:1981.82 1:24:56 81.24 1:30:33 79.20 1:36:10 79.14 1:41:47 78.47 1:47:2477.88 1:53:02 78.29 1:58:39 78.56 2:04:16 77.21 2:21:12 76.06 2:26:5077.34 2:49:21 75.34 3:17:30 72.37 3:51:16 50.13 4:19:26 51.88 4:47:3848.95 5:21:25 45.19 5:49:35 47.44 6:17:45 44.94 6:51:31 43.29 7:19:4041.85 7:47:22 41.72 8:21:11 36.89 8:49:19 37.52 9:17:30 36.34 9:51:1734.61 10:19:29  31.94 10:47:39  32.33 11:15:53  29.85 11:49:44  29.9412:17:54  27.99 12:46:10  27.39 13:20:01  26.14

Conclusion:

In simulated gastric fluid at 37° C., ER-876400 was found to degrade by50% in less than 30 seconds while ER-876437 has a half-life of roughly4-6 hours.

Example V Effect of ER-876437 on a Non-Decitabine CDA Substrate inSurvival Murine Lymphoma L1210 Model

This study may be employed to determine whether ER-876437 enhances theoral efficacy of a non-decitabine CDA substrate (or prodrug thereof) inthe L1210 survival model in mice.

Preparation of L1210 Cells:

L1210 ascitic cells may be prepared by passaging them in mice at leastthree times as follows. Each CD2F1 female mouse may be intraperitoneally(IP) injected with about 10⁵ L1210 ascitic cells. After one week, themouse may be sacrificed (asphyxiation via CO₂). After sacrificing, themouse may be placed on its back, its belly surface may be cleaned withalcohol wipes, and a small incision may be made into the peritonealcavity. 2 ml of ice cold 2.1% BSA in saline may be injected into thecavity and then the fluid may be withdrawn and transferred with an 18G 3cc syringe into a clean sterile tube and kept on ice. The fluid may bediluted 1:10 in 2.1% BSA in saline and one drop of Zap oglobin II lyticreagent (available from Beckman Coulter, Inc.) may be added to 1 ml ofdiluted ascites. Diluted ascites (diluted 1:10 again) may be counted ona hematocytometer and the number of cells per mL may be calculated.About 10⁵ L1210 cells may be used for a subsequent passage for anothermouse passage. Or, a stock of L1210 ascites in BSA solution may bediluted to 1×10⁴ cells/0.1 ml for use in the study mice.

Preparation of Study Mice:

CD2F1 6-7 weeks old female mice may be randomly separated into groupssuch as those identified in Table 8. The mice may be prepared withintravenous (IV) injection of L1210 ascites (prepared as describedabove) one day prior to commencing the dosing. Mice may be injected with0.1 ml of cell solution via caudal vein with a 27 G needle.

Mice may be dosed with vehicle or ER-876437 per os (PO, i.e., orally) 30minutes prior to dosing with non-decitabine CDA substrate. ER-876437 maybe prepared at 1 mg/ml in PBS and then diluted to 0.1 mg/ml, 0.01 mg/mland 0.001 mg/ml in PBS for the lower doses.

A non-decitabine CDA substrate may be prepared at a 1 mg/ml stock in PBSand appropriately diluted to achieve a 0.01 mg/ml dosing solution.ER-876437 may be prepared at the beginning of each day of dosing andstored at 4° C. The non-decitabine CDA substrate may be prepared freshtwice a day, just prior to dosing. All solutions may be stored on icewhile dosing. Mice may be dosed (intraperitoneally (IP) or per os(orally, PO)) twice a day (8 hours apart) for 4 consecutive days. Aproposed final dosing scheme and proposed total non-decitabine CDAsubstrate (NDCS) and ER-876437 dose is outlined in Table 8. In theproposed dosing scheme, mice may be dosed (with vehicle, ER-876437 orNDCS) orally, intraperitoneally, or intravenously.

TABLE 8 Proposed Dosing Scheme NDCS Cumulative ER- Cumulative Group DoseNDCS 876437 ER-876437 # Drug (rte Adm) Dose Dose Dose 1 Vehicle Veh 0mg/kg Veh 0 mg/kg 2 ER-876437 Veh 0 mg/kg 10 mg/kg 80 mg/kg 3 NDCS 0.1mg/kg 0.8 mg/kg Veh 0 mg/kg 4 NDCS/ER- 0.1 mg/kg 0.8 mg/kg 0.01 mg/kg0.08 mg/kg 876437 5 NDCS/ER- 0.1 mg/kg 0.8 mg/kg 0.1 mg/kg 0.8 mg/kg876437 6 NDCS/ER- 0.1 mg/kg 0.8 mg/kg 1 mg/kg 8 mg/kg 876437 7 NDCS/ER-0.1 mg/kg 0.8 mg/kg 1 0 mg/kg 80 mg/kg 876437

Survival and Autopsy:

Mice may be observed for survival and weighed daily for the duration ofthe study (30 days). Dead mice may be autopsied and observed for thepresence of tumors in organs. Tumor deaths may be determined by liverweights greater than 1.6 g and spleen weights greater than 150 mg as perCovey J M and Zaharko D S, Eur J Cancer Clin Oncol, Vol. 21 p. 109-117,1985.

Conclusions regarding whether co-administration of ER-876437 with anon-decitabine CDA substrate enhances survival as compared toadministration of the non-decitabine CDA substrate alone in the L1210survival model in mice may then be determined from the resulting data.

Example VI In Vivo Efficacy Study of ER-876437 and Gemcitabine in A2780Human Ovarian Cancer Xenograft Model

This study evaluated the enhancing activity of ER-876437 on oralgemcitabine treatment in an A2780 human ovarian cancer xenograft model.ER-876437 was dosed 30 minutes prior to gemcitabine and both compoundswere dosed orally. Animals were dosed daily from Monday to Friday fortwo weeks.

Materials and Methods

ER-876437 and gemcitabine-HCl (Gemzar® injectable, Eli Lilly) wereformulated into 0.5% methyl cellulose (Sigma). Female nude mice (NU/NU,strain code 088, 6 weeks old, Charles River Laboratory) were implantedsubcutaneously with 5×10⁶ A2780 cancer cells per mouse. On day 13 whenthe tumors were approximately 150 mm³, treatment started as described inTable 9.

TABLE 9 Dosing Scheme for gemcitabine and ER-876437 gemcitabineER-876437* (PO, qdx5 for (PO, qdx5 for Group Treatment two weeks) twoweeks) 1 vehicle (0.5% methyl cellulose) 2 gemcitabine 1 mg/kg 3ER-876437 10 mg/kg 4 ER-876437*/gemcitabine 1 mg/kg 10 mg/kg *ER-876437was dosed approximately 30 minutes prior to gemcitabine

Tumor volume and regressions were followed over time. Tumor volume wascalculated by (length×width²)/2. Note that a complete regression wasdefined as no measurable tumor for at least 3 consecutive measurements;while a partial regression was defined as tumor shrinkage to equal orless than 50% of original tumor volume for 3 consecutive measurements.Tumor growth delay (TGD) was defined as the median number of days forthe control and treatment groups to grow to 342.14 mm³. The averagetumor volume on the first day of treatment (day 13) is 171.07 mm³.Hence, twice as much as the initial tumor size is 342.14 mm³.

Results:

ER-876437 alone (Group 3) had no effect at all on tumor growth (FIG. 3).Oral administration of gemcitabine in the regimen of 1 mg/kg PO qdx5 fortwo weeks (Group 2) showed limited efficacy after second week oftreatment (FIG. 3), while ER-876437 alone (Group 3) did not show anyefficacy during the whole treatment period (FIG. 3). When tumor doublingtime is used to define the tumor growth delay (TGD), both gemcitabinealone (Group 2) and ER-876437 alone (Group 3) showed merely 2 days delayas compared to vehicle (Group 1) (Table 10). There is no statisticallysignificant difference amongst Groups 1, 2 and 3 (Mann-Whitney test,GraphPad Prism 5, La Jolla, Calif.), with no regressions or tumor freesurvivors at day 41.

In contrast, when ER-876437 was administered approximately 30 minutesprior to gemcitabine (Group 4), one out of 10 mice (10%) showed completeregression and was a tumor free survivor at the study termination day(day 41). 3 out of 10 mice (30%) also showed partial tumor regression.These results show that there is therapeutic efficacy observed in theER-876437/gemcitabine combination (Group 4) as compared to vehicle(Group 1), or as compared to gemcitabine alone (Group 2) (FIG. 3).Significant difference in TGD is observed when comparing thiscombination (Group 4) to gemcitabine alone (Group 2) (P=0.0001,Mann-Whitney test, GraphPad Prism 5, La Jolla, Calif., Table 10).

TABLE 10 Effect of Combination Treatment of Oral Gemcitabine and OralER-876437 on Tumor Growth Delay in the A2780 Ovarian Cancer Model.Treatment TGD^(†) P value* Vehicle NA 1 mg/kg gemcitabine 2 days 10mg/kg ER-876437 2 days 1 mg/kg gemcitabine + 23 days 0.0001 10 mg/kgER-876437 Note: ^(†)TGD, tumor growth delay. *Mann-Whitney test was usedto assess whether tumor growth delay differ significantly betweengemcitabine alone group and ER-876437 plus gemcitabine combinationgroup.Conclusion:

Pre-treatment of ER-876437 showed significant enhancement of therapeuticactivity of oral gemcitabine in this study. Significant tumor growthdelay in the combination group compared to oral gemcitabine alone wasidentified with the Mann-Whitney statistical test (GraphPad Prism 5, LaJolla, Calif.).

Example VII Effect of ER-876437 on the Half-Life of Gemcitabine in thePresence of CDA in Tris-HCl Buffer at 37° C.

This example describes the effect of ER-876437 on the half-life(T_(1/2)) of gemcitabine in the presence of cytidine deaminase (CDA) inTris-HCl buffer at 37° C.

Materials and Equipment

This Example employed a Phenomenex Luna C18(2) HPLC column (100 Å4.6×250 mm 5 μm). The solvent delivery system employed an HPLCquaternary pump, low pressure mixing. An autosampler having a variableloop, 0.1 to 100 μL range and temperature controlled thermostat wasused. The UV detector can employ a dual wavelength detector, a diodearray detector, a variable wavelength detector or equivalent, and can berecorded using chromatographic software (e.g., Waters Empower 2 Build2154, Agilent ChemStation software version A.09.03 or higher for HPLC orequivalent). The analytical balance employed was capable of weighing±0.1 mg. Degassed HPLC grade water and degassed HPLC grade acetonitrilewere used as solvents for the mobile phases.

Diluting solution used to make the below solutions was Tris-HCl (37° C.,ph 7.4, Boston BioProducts). Diluting solution also served as the blankfor the UV spectra.

Gemcitabine Standard Control:

0.2 mM gemcitabine control was prepared by weighing 2.6 mg ofgemcitabine in a 10 mL volumetric flask. The flask was diluted to volumewith Tris-HCl buffer stored at 37° C. and mixed by inversion. Solutionwas labeled as gemcitabine stock solution. 1.0 mL of gemcitabine stocksolution was transferred to a 5 mL volumetric flask and diluted tovolume with the diluting solution and mixed by inversion.

ER-876437 Standard Control:

0.4 mM ER-876437 control was prepared by weighing 5.2 mg of ER-876437 ina 10 mL volumetric flask. The flask was diluted to volume with Tris-HClbuffer stored at 37° C. and mixed by inversion. Solution was labeled asER-876437 stock solution. 1.0 mL of ER-876437 stock solution wastransferred to a 5 mL volumetric flask and diluted to volume with thediluting solution and mixed by inversion.

Gemcitabine with CDA:

1.0 mL of gemcitabine stock solution was transferred to a 5 mLvolumetric flask. Approximately 2-3 mL of diluting solution wastransferred to the flask. 0.125 mL of CDA solution was transferred tothe flask and diluted to volume with diluting solution. The sample wasmixed by inversion and injected into the HPLC immediately afterpreparing.

Gemcitabine with CDA and ER-876437:

1.0 mL of ER-876437 stock solution was transferred to a 5 mL volumetricflask. Approximately 2 mL of diluting solution was transferred to theflask. 0.125 mL of CDA solution was transferred to the flask. 1.0 mL ofgemcitabine stock solution was transferred to the same flask and dilutedto volume with diluting solution. The sample was mixed by inversion andinjected into the HPLC immediately after preparing.

HPLC Parameters:

The above solutions were run on an HPLC column using the parametersshown in Table 11.

TABLE 11 HPLC Parameters Column Temperature: 25° C. AutosamplerTemperature: 37° C. Flow rate: 1.0 mL/min. Flow rate may be adjusted±0.2 mL/min to obtain specified retention times. Time, %-Solvent%-Solvent Gradient: min A* B* Initial 96  4 10 96  4 20 75 25 25 75 25Re-equilibration time 10 minutes Injection volume: 25 μL Needle WashSolution: Use the diluting solution Detection: 205 nm UV Run Time: 25minutes *Solvent A: water; solvent B: acetonitrile

The retention time for gemcitabine was found to be approximately 8minutes; and the retention time of ER-876437 was found to beapproximately 21.8 minutes.

Results and Discussion

TABLE 12 Summary of Results Solutions Estimated T_(1/2) Gemcitabine withCDA in Tris-HCl buffer at 37° C. <35 minutes Gemcitabine with CDA andER-876437 in Tris-HCl More than 13 h buffer at 37° C.

The levels of gemcitabine, in the presence and absence of CDA, with orwithout ER-876437, in Tris-HCl buffer at 37° C. were measured by HPLCanalysis using UV detection. The areas of the gemcitabine and ER-876437peaks in the experimental samples were measured and compared to theareas of the gemcitabine and ER-876437 time zero injections,respectively. Results were reported as percent remaining of control.Data was collected at 205 nm UV because gemcitabine and ER-876437 sharethis UV maximum. See FIG. 4. Results were captured every 35 minutes for12 hours and intermittently thereafter due to the length of theanalytical method. HPLC chromatograms showing overlaid traces atspecified time points are shown in FIG. 5 and FIG. 6.

HPLC chromatograms in these figures are shown with a constant, additiveoffset for clarity. Although the bottom trace is shown starting attime=0.00 minutes, each successive chromatogram is arbitrarily shiftedto the right of the previous chromatogram (by a constant amount of time)so as to avoid having the peaks overlap. The actual times associatedwith the peaks shown in these chromatograms can be realized by shiftingthe start of the chromatogram trace (at the left hand side) back to thevertical axis where time equals 0.00 minutes. Similarly, the actual UVabsorption of any peak can be realized by shifting the baseline of thechromatogram to the position where mAU=0.00.

In the absence of CDA, no reduction in the gemcitabine concentration wasobserved after 10 hours, while, in the presence of CDA, theconcentration of gemcitabine was reduced to nearly 0% control within 1hour and the T_(1/2) was found to be <35 minutes. Addition of ER-876437to the incubation mixture resulted in inhibition of the reaction withgreater than 95% of gemcitabine remaining after 7 h. Similarly, thelevels of ER-876437 were not affected after 7 hours of exposure to CDAwith gemcitabine. A summary of all the results are shown in FIG. 7.

In conclusion, the T_(1/2) of gemcitabine in the presence of CDA inTris-HCl buffer at 37° C. was found to be <35 minutes. ER-876437 nearlycompletely inhibited this effect. Gemcitabine alone in Tris-HCl bufferat 37° C. did not show any degradation at the end of observation

Example VIII Effect of ER-876437 on the Half-Life of Cytarabine in thePresence of CDA in Tris-HCl Buffer at 37° C.

This example describes the effect of ER-876437 on the half-life(T_(1/2)) of cytarabine (Sigma) in the presence of cytidine deaminase(CDA) in Tris-HCl buffer at 37° C.

With exceptions identified below, materials and equipment are the sameas were described above for Example VII.

Diluting solution used to make the below solutions was Tris-HCl (37° C.,ph 7.4, Boston BioProducts). Diluting solution also served as the blankfor the UV spectra.

Cytarabine Standard Control: 0.2 mM cytarabine control was prepared byweighing 2.4 mg of cytarabine in a 10 mL volumetric flask. The flask wasdiluted to volume with Tris-HCl buffer stored at 37° C. and mixed byinversion. Solution was labeled as cytarabine stock solution. 1.0 mL ofcytarabine stock solution was transferred to a 5 mL volumetric flask anddiluted to volume with the diluting solution and mixed by inversion.

ER-876437 Standard Control: 0.4 mM ER-876437 control was prepared byweighing 5.2 mg of ER-876437 in a 10 mL volumetric flask. The flask wasdiluted to volume with Tris-HCl buffer stored at 37° C. and mixed byinversion. Solution was labeled as ER-876437 stock solution. 1.0 mL ofER-876437 stock solution was transferred to a 5 mL volumetric flask anddiluted to volume with the diluting solution and mixed by inversion.

Cytarabine with CDA:

1.0 mL of cytarabine stock solution was transferred to a 5 mL volumetricflask. Approximately 2-3 mL of diluting solution was transferred to theflask. 0.125 mL of CDA solution was transferred to the flask and dilutedto volume with diluting solution. The sample was mixed by inversion andinjected into the HPLC immediately after preparing.

Cytarabine with CDA and ER-876437: 1.0 mL of ER-876437 stock solutionwas transferred to a 5 mL volumetric flask. Approximately 2 mL ofdiluting solution was transferred to the flask. 0.125 mL of CDA solutionwas transferred to the flask. 1.0 mL of cytarabine stock solution wastransferred to the same flask and diluted to volume with dilutingsolution. The sample was mixed by inversion and injected into the HPLCimmediately after preparing.

The above standards and samples were run on an HPLC column using theparameters shown in Table 11 of Example VII, except that UV spectra werecollected at 205 and 275 nm. The retention time for cytarabine was foundto be approximately 4.4 minutes; and the retention time of ER-876437 wasfound to be approximately 21.8 minutes.

Results and Discussion

TABLE 13 Summary of Results Solutions Estimated T_(1/2) Cytarabine withCDA in Tris-HCl buffer at 37° C. <35 minutes Cytarabine with CDA andER-876437 in Tris-HCl More than 52 h buffer at 37° C.

The levels of cytarabine, in the presence and absence of CDA, with orwithout ER-876437, in Tris-HCl buffer at 37° C. were measured by HPLCanalysis using UV detection. The areas of the cytarabine and ER-876437peaks in the stability samples were measured and compared to the areasof the cytarabine and ER-876437 standard controls, respectively. Resultswere reported as percent remaining of control.

Since ER-876437 and cytarabine have different UV maxima, HPLCchromatograms were collected at 205 nm and 275 nm UV. Cytarabine resultswere calculated using 275 nm UV and ER-876437 results were calculatedusing 205 nm UV. See FIG. 8 for ER-876437 and cytarabine UV Spectra.

Results were captured every 35 minutes for 12 hours and intermittentlythereafter due to the length of the analytical method. HPLCChromatograms showing overlaid traces at specified time points are shownin FIGS. 9 and 10.

HPLC chromatograms in these figures are shown with a constant, additiveoffset for clarity. Although the bottom trace is shown starting attime=0.00 minutes, each successive chromatogram is arbitrarily shiftedto the right of the previous chromatogram (by a constant amount of time)so as to avoid having the peaks overlap. The actual times associatedwith the peaks shown in these chromatograms can be realized by shiftingthe start of the chromatogram trace (at the left hand side) back to thevertical axis where time equals 0.00 minutes. Similarly, the actual UVabsorption of any peak can be realized by shifting the baseline of thechromatogram to the position where mAU=0.00.

In the absence of CDA, no reduction in the cytarabine concentration wasobserved after 55 hours, while, in the presence of CDA, theconcentration of cytarabine was reduced to nearly 0% control within 35minutes and the T_(1/2) was found to be <35 minutes. Addition ofER-876437 to the incubation mixture resulted in inhibition of thereaction with greater than 95% of cytarabine remaining after 52 h.Similarly, the levels of ER-876437 were not affected after 52 hours ofexposure to CDA with cytarabine. A summary of all the results are shownin FIGS. 11 and 12.

In conclusion, the T_(1/2) of cytarabine in the presence of CDA inTris-HCl buffer at 37° C. was found to be <35 minutes. ER-876437 nearlycompletely inhibited this effect. Cytarabine alone in Tris-HCl buffer at37 C did not show any degradation at the end of observation period (52h).

Example IX Effect of Er-876437 on Decitabine in Survival Murine LymphomaL1210 Model

The purpose of this study was to determine if ER-876437 enhances theoral efficacy of decitabine in the L1210 survival model in mice.

Preparation of L1210 Cells:

L1210 ascitic cells were prepared by passaging them in mice at leastthree times as follows. Each CD2F1 female mouse was intraperitoneally(IP) injected with about 10⁵ L1210 ascitic cells. After one week, themouse was sacrificed (asphyxiation via CO₂). The mouse was placed on itsback, its belly surface was cleaned with alcohol wipes, and a smallincision was made into the peritoneal cavity. 2 ml of ice cold 2.1% BSAin saline was injected into the cavity and then the fluid was withdrawnand transferred with an 18 G 3 cc syringe into a clean sterile tube andkept on ice. The fluid was diluted 1:10 in 2.1% BSA in saline and onedrop of Zap o globin II lytic reagent (available from Beckman Coulter,Inc.) was added to 1 ml of diluted ascites. Diluted ascites (diluted1:10 again) were counted on a hematocytometer and the number of cellsper mL was calculated. About 10⁵ L1210 cells were used for a subsequentpassage for another mouse passage. Or, a stock of L1210 ascites in BSAsolution was diluted to 1×10⁴ cells/0.1 ml for use in the study mice.

Preparation of Study Mice:

35 CD2F1 6-7 weeks old female mice were randomly separated into the 7groups identified in Table 14. The mice were prepared with intravenous(IV) injection of L1210 ascites (prepared as described above) one dayprior to commencing the dosing. Mice were injected with 0.1 ml of cellsolution via caudal vein with a 27 G needle. The total IV injection forall mice took about 50 minutes.

Mice were dosed with vehicle or ER-876437 per os (PO, i.e., orally) 30minutes prior to dosing with decitabine. ER-876437 was prepared at 1mg/ml in PBS and then diluted to 0.1 mg/ml and 0.01 mg/ml in PBS for thelower doses.

Decitabine was prepared at a 1 mg/ml stock in PBS and appropriatelydiluted to achieve a 0.01 mg/ml dosing solution. ER-876437 was preparedat the beginning of each day of dosing and stored at 4° C. Decitabinewas prepared fresh twice a day, just prior to dosing. All solutions werestored on ice while dosing. Mice were dosed (intraperitoneally (IP) orper os (orally, PO)) twice a day (8 hours apart) for 4 consecutive days.Final dosing scheme and total decitabine and ER-876437 dose is outlinedin Table 14.

TABLE 14 Dosing Scheme decitabine Cumulative ER- Cumulative Group Dosedecitabine 876437 ER-876437 # Drug (rte Adm) Dose Dose Dose 1 VehicleVeh 0 mg/kg Veh 0 mg/kg 2 ER-876437 Veh 0 mg/kg 10 mg/kg 80 mg/kg 3decitabine 0.1 mg/kg 0.8 mg/kg Veh 0 mg/kg PO 4 decitabine/ER- 0.1 mg/kg0.8 mg/kg 0.1 mg/kg 0.8 mg/kg 876437 PO 5 decitabine/ER- 0.1 mg/kg 0.8mg/kg 1 mg/kg 8 mg/kg 876437 PO 6 decitabine/ER- 0.1 mg/kg 0.8 mg/kg 10mg/kg 80 mg/kg 876437 PO 7 decitabine 0.1 mg/kg 0.8 mg/kg Veh 0 mg/kg IP

Survival and Autopsy:

Mice were observed for survival and weighed daily (Mon-Fri) for theduration of the study (30 days). Dead mice were autopsied and observedfor the presence of tumors in organs. Tumor deaths were determined byliver weights greater than 1.6 g and spleen weights greater than 150 mgas per Covey J M and Zaharko D S, Eur J Cancer Clin Oncol, Vol. 21 p.109-117, 1985.

Results:

Mice dosed with decitabine and decitabine plus ER-876437 lived longerthan vehicle controls and ER-876437 alone (Table 15 and 16; p<0.05). Nodose response was observed with ER-876437 in combination withdecitabine.

0.1 mg/kg decitabine PO was slightly less effective than 0.1 mg/kgdecitabine IP (Tables 15 and 16; p=0.0047). Co-administration ofER-876437 with 0.1 mg/kg decitabine regardless of ER-876437 dosesignificantly enhanced survival (days) compared to 0.1 mg/kg decitabinePO or IP (Tables 15 and 16; p<0.05), but there was no dose responsebetween ER-876437 doses.

Table 15 lists the mean survival of each treatment group and the percentILS (increased life span) compared to the vehicle group. All treatedgroups lived significantly longer than vehicle controls and CDAinhibitor alone groups (p<0.05).

Listed in Table 15 are the weight of the livers and spleens of mice onautopsy. All mice died a ‘tumor burden’ related death as indicated bythe liver weights greater than 1.6 gram and the spleen weights greaterthan 150 mg (Covey et al Eur J Cancer Oncol 1985).

Gross observations were noted concerning the overall appearance of theperitoneal and thoracic cavities; however, there were no formal analysisof these observations.

TABLE 15 Effect of decitabine and ER-876437 on survival and liver andspleen weights in the L1210 IV Survival Model % ILS Mean Survival(Increased Mean Liver Mean Spleen Group (days) ± SD Life Span) wts (g) ±SD wts (g) ± SD Veh/Veh  7.4 ± 0.55 1.79 ± 0.34 0.35 ± 0.03Veh/decitabine 0.1 mg/kg PO 11.2 ± 0.45 51.35 2.17 ± 0.1  0.34 ± 0.04Veh/decitabine 0.1 mg/kg IP 13.4 ± 0.89 81.08 1.81 ± 0.25 0.37 ± 0.14ER-876437 0.1 mg/kg/decitabine 0.1 mg/kg PO 15.8 ± 1.48 113.51 1.92 ±0.16 0.24 ± 0.04 ER-876437 1 mg/kg/decitabine 0.1 mg/kg PO 16.6 ± 1.52124.32  2.2 ± 0.46 0.28 ± 0.13 ER-876437 10 mg/kg/decitabine 0.1 mg/kgPO 17.2 ± 2.39 132.43 2.18 ± 0.31 0.38 ± 0.12 ER-876437 10 mg/kg/Veh 7.6 ± 0.89 2.70 2.02 ± 0.07 0.37 ± 0.04${*\mspace{14mu}\%\mspace{14mu}{ILS}} = \frac{{{mean}\mspace{14mu}{survival}\mspace{14mu}{of}\mspace{14mu}{experimental}\mspace{14mu}({days})} - {{mean}\mspace{14mu}{survival}\mspace{14mu}{controls}\mspace{14mu}({days}) \times 100}}{{Mean}\mspace{14mu}{survival}\mspace{14mu}{of}\mspace{14mu}{control}\mspace{14mu}({days})}$

TABLE 16 Statistical Analysis (Log Rank Test as per Prism GraphPad)Comparison P value Control vs 0.1 mg/kg decitabine PO 0.0023 Control vs0.1 mg/kg decitabine IP 0.0023 Control vs ER-876437 alone 0.601decitabine 0.1 mg/kg PO vs. decitabine 0.1 mg/kg IP 0.0047 Control vsER-876437 0.1 mg/kg/decitabine 0.1 mg/kg PO 0.0023 decitabine 0.1 mg/kgPO vs. ER-876437 0.1 mg/kg/ 0.0016 decitabine 0.1 mg/kg PO decitabine0.1 mg/kg PO vs. ER-876437 1 mg/kg/ 0.0016 decitabine 0.1 mg/kg POdecitabine 0.1 mg/kg PO vs. ER-876437 10 mg/kg/ 0.0016 decitabine 0.1mg/kg PO decitabine 0.1 mg/kg IP vs. ER-876437 0.1 mg/kg/ 0.0119decitabine 0.1 mg/kg PO decitabine 0.1 mg/kg IP vs. ER-876437 1 mg/kg/0.0034 decitabine 0.1 mg/kg PO decitabine 0.1 mg/kg IP vs. ER-876437 10mg/kg/ 0.0034 decitabine 0.1 mg/kg PO ER-876437 0.1 mg/kg/decitabine 0.1mg/kg PO vs. 0.4069 ER-876437 1 mg/kg/decitabine 0.1 mg/kg PO ER-8764371 mg/kg/decitabine 1 mg/kg PO vs. 0.6131 ER-876437 10 mg/kg/decitabine0.1 mg/kg POConclusion:

Decitabine plus ER-876437 was more efficacious in the L1210 IV survivalmodel than decitabine alone regardless of the route administration ofdecitabine (PO or IP). There was no dose response between 0.1 mg/kg, 1mg/kg and 10 mg/kg of ER-876437 plus decitabine groups. This experimentwas repeated in Example VI but using lower doses of ER-876437 todetermine the minimally effective dose.

Example X Effect of ER-876437 on Decitabine in Survival Murine LymphomaL1210 Model

This example followed all the methods and protocols of Example VIII withthe following changes: 40 CD2F1 6-7 weeks old female mice were randomlyseparated into the 8 groups identified in Table 17. Preparation of studymice with IV injection of L1210 ascites took about 60 minutes. ER-876437was prepared at 1 mg/ml in PBS and then diluted to 0.1 mg/ml, 0.01 mg/mland 0.001 mg/ml in PBS. All solutions were stored on ice while dosing.Mice were dosed (IP or PO) twice a day (7 or 8 hours apart) for 4consecutive days.

TABLE 17 Dosing Scheme Decitabine Cumulative ER- ER- Group DoseDecitabine 876437 876437 # Drug (rte Adm) Dose Dose Dose 1 VehicleVehicle 0 mg/kg Vehicle 0 mg/kg 2 ER-876437 Vehicle 0 mg/kg 1 mg/kg 8mg/kg 3 decitabine 0.1 mg/kg 0.8 mg/kg Veh 0 mg/kg PO 4 decitabine/ER-0.1 mg/kg 0.8 mg/kg 0.01 mg/kg 0.08 mg/kg 876437 PO 5 decitabine/ER- 0.1mg/kg 0.8 mg/kg 0.1 mg/kg 0.8 mg/kg 876437 PO 6 decitabine/ER- 0.1 mg/kg0.8 mg/kg 1 mg/kg 8 mg/kg 876437 PO 7 decitabine 0.1 mg/kg 0.8 mg/kgVehicle 0 mg/kg IP  8* decitabine 0.1 mg/kg 0.8 mg/kg Vehicle 0 PO*Dosed twice a day, 8 hours apart. All other groups were dosed twice aday, 7 hours apart.Results:

Mice dosed with decitabine and decitabine plus ER-876437 lived longerthan vehicle controls and ER-876437 alone (Tables 18 and 19; p<0.05).There was no difference in survival of mice dosed with 0.1 mg/kgdecitabine PO when dosed 7 or 8 hours apart (Tables 18 and 19).

0.1 mg/kg decitabine PO was less effective than 0.1 mg/kg decitabine IP(Tables 18 and 19; p=0.0086). Co-administration of 0.01 mg/kg ER-876437with 0.1 mg/kg decitabine PO had no effect on extending survival inL1210 leukemic mice compared to decitabine PO alone. Co-administrationof 0.1 mg/kg and 1 mg/kg ER-876437 with 0.1 mg/kg decitabine POsignificantly enhanced survival (days) compared to 0.1 mg/kg decitabinePO alone. Co-administration of 0.1 mg/kg and 1 mg/kg with 0.1 mg/kgdecitabine PO alone was not statistically different than 0.1 mg/kgdecitabine administered via IP. ER-876437 had a slight dose response atthese low doses: 0.01 mg/kg was ineffective at enhancing the effect ofdecitabine delivered orally while both 0.1 mg/kg and 1 mg/kgsignificantly enhanced survival (p=0.04 and p=0.005 respectively; Table19). The two higher doses of ER-876437 in combination with decitabine POhad a slight dose response (p=0.09; Table 19).

Table 18 lists the mean survival of each treatment group and the percentILS (increased life span) compared to the vehicle group. All treatedgroups live significantly longer than vehicle controls and groups givenER-876437 only (p<0.05).

Listed in Table 18 are the weight of the livers and spleens of mice onautopsy. All mice died a ‘tumor burden’ related death as indicated bythe liver weights greater than 1.6 gram and the spleen weights greaterthan 150 mg (Covey J M and Zaharko D S, Eur J Cancer Clin Oncol, Vol. 21p. 109-117, 1985).

Gross observations were noted concerning the overall appearance of theperitoneal and thoracic cavities; however, there were no formal analysisof these observations.

TABLE 18 Effect of Decitabine and ER-876437 on survival and liver andspleen weights in the L1210 IV Survival Model % ILS Mean Survival(Increased Mean Liver Mean Spleen Group (days) ± SD Life Span) wts (g) ±SD wts (g) ± SD Veh/Veh   8 ± 0.71 1.95 ± 0.15 0.35 ± 0.03Veh/decitabine 0.1 mg/kg PO 11.8 ± 0.84 47.50 2.02 ± 0.31 0.36 ± 0.11Veh/decitabine 0.1 mg/kg PO (8 hr.) 11.6 ± 0.55 45.00 1.81 ± 0.41 0.33 ±0.06 Veh/decitabine 0.1 mg/kg IP 13.6 ± 0.55 70.00 2.01 ± 0.41 0.31 ±0.07 ER-876437 0.01 mg/kg/decitabine 0.1 mg/kg PO  12 ± 0.0 50.00 2.06 ±0.23 0.32 ± 0.04 ER-876437 0.1 mg/kg/decitabine 0.1 mg/kg PO 13.2 ± 0.8465.00 2.28 ± 0.25 0.35 ± 0.1  ER-876437 1 mg/kg/decitabine 0.1 mg/kg PO14.2 ± 0.84 77.50 2.24 ± 0.32 0.34 ± 0.09 ER-876437 1.0 mg/kg/Veh  8.4 ±0.55 5.00 2.04 ± 0.15 0.32 ± 0.02 Veh: vehicle only${*\mspace{14mu}\%\mspace{14mu}{ILS}} = \frac{{{mean}\mspace{14mu}{survival}\mspace{14mu}{of}\mspace{14mu}{experimental}\mspace{14mu}{group}\mspace{14mu}({days})} - {{mean}\mspace{14mu}{survival}\mspace{14mu}{controls}\mspace{14mu}{group}\mspace{14mu}({days}) \times 100}}{{Mean}\mspace{14mu}{survival}\mspace{14mu}{of}\mspace{14mu}{control}\mspace{14mu}{group}\mspace{14mu}({days})}$

TABLE 19 Statistical Analysis (Log Rank Test as per Prism GraphPad)Comparison P value Control vs 0.1 mg/kg decitabine PO 0.002 Control vs0.1 mg/kg decitabine PO (8 hr.) 0.002 Control vs 0.1 mg/kg decitabine IP0.002 Control vs ER-876437 alone 0.353 Decitabine 0.1 mg/kg PO vs.decitabine 0.1 mg/kg PO 0.6015 (8 hr.) decitabine 0.1 mg/kg PO vs.decitabine 0.1 mg/kg IP 0.0086 Control vs ER-876437 0.01 mg/kg/ 0.002decitabine 0.1 mg/kg PO decitabine 0.1 mg/kg PO vs. ER-876437 0.1 mg/kg/0.649 decitabine 0.1 mg/kg PO decitabine 0.1 mg/kg PO vs. ER-876437 0.1mg/kg/ 0.0368 decitabine 0.1 mg/kg PO decitabine 0.1 mg/kg PO vs.ER-876437 1 mg/kg/ 0.0048 decitabine 0.1 mg/kg PO decitabine 0.1 mg/kgIP vs. ER-876437 1 mg/kg/ 0.1729 decitabine 0.1 mg/kg PO ER-876437 0.01mg/kg/decitabine 0.1 mg/kg PO vs. 0.014 ER-876437 0.1 mg/kg/decitabine0.1 mg/kg PO ER-876437 0.1 mg/kg/decitabine 1 mg/kg PO vs. 0.0889ER-876437 1 mg/kg/decitabine 0.1 mg/kg POConclusion:

Co-administration of ER-876437 at both 0.1 mg/kg and 1 mg/kg with 0.1mg/kg decitabine PO enhanced survival compared to decitabineadministered PO alone but not decitabine administered IP in the L1210survival model in mice. The lowest dose tested (0.01 mg/kg) had noeffect on enhancing survival of mice treated with 0.1 mg/kg decitabinewhen administered via PO. The two higher doses of ER-876437 incombination with decitabine had a slight dose response (p=0.09). Theminimally effective dose of ER-976437 in this model was found to be 0.1mg/kg.

There was no difference in survival of mice dosed with 0.1 mg/kgdecitabine PO 2×/day qd×4 7 hours apart or 8 hours apart.

All publications or patent applications described herein are herebywholly incorporated by reference.

Example XI Effect of ER-876437 on the Half-Life of Decitabine in thePresence of CDA in Tris-HCl Buffer at 37° C.

This example describes the effect of ER-876437 on the half-life (T₁₁₂)of decitabine (Eisai) in the presence of cytidine deaminase (CDA) inTris-HCl buffer at 37° C.

Materials and Equipment

This Example employed a Phenomenex Luna C18(2) HPLC column (100 Å4.6×250 mm 5 μm). The solvent delivery system employed an HPLCquaternary pump, low pressure mixing. An autosampler having a variableloop, 0.1 to 100 μL range and temperature controlled thermostat wasused. The UV detector can employ a dual wavelength detector, a diodearray detector, a variable wavelength detector or equivalent, and can berecorded using chromatographic software (e.g., Waters Empower 2 Build2154, Agilent ChemStation software version A.09.03 or higher for HPLC orequivalent). The analytical balance employed was capable of weighing±0.1 mg. Degassed HPLC grade water and degassed HPLC grade acetonitrilewere used as solvents for the mobile phases.

Diluting solution used to make the below solutions was Tris-HCl (37° C.,ph 7.4, Boston BioProducts). Diluting solution also served as the blankfor the UV spectra.

Decitabine Standard Control: 0.2 mM decitabine control was prepared byweighing 2.6 mg of decitabine in a 10 mL volumetric flask. The flask wasdiluted to volume with Tris-HCl buffer stored at 37° C. and mixed byinversion. Solution was labeled as decitabine stock solution. 1.0 mL ofdecitabine stock solution was transferred to a 5 mL volumetric flask anddiluted to volume with the diluting solution and mixed by inversion.

ER-876437 Standard Control: 0.4 mM ER-876437 control was prepared byweighing 5.2 mg of ER-876437 in a 10 mL volumetric flask. The flask wasdiluted to volume with Tris-HCl buffer stored at 37° C. and mixed byinversion. Solution was labeled as ER-876437 stock solution. 1.0 mL ofER-876437 stock solution was transferred to a 5 mL volumetric flask anddiluted to volume with the diluting solution and mixed by inversion.

Decitabine with CDA 1.0 mL of decitabine stock solution was transferredto a 5 mL volumetric flask. Approximately 2-3 mL of diluting solutionwas transferred to the flask. 0.125 mL of CDA solution was transferredto the flask and diluted to volume with diluting solution. The samplewas mixed by inversion and injected into the HPLC immediately afterpreparing.

Decitabine with CDA and ER-876437: 1.0 mL of ER-876437 stock solutionwas transferred to a 5 mL volumetric flask. Approximately 2 mL ofdiluting solution was transferred to the flask. 0.125 mL of CDA solutionwas transferred to the flask. 1.0 mL of decitabine stock solution wastransferred to the same flask and diluted to volume with dilutingsolution. The sample was mixed by inversion and injected into the HPLCimmediately after preparing.

HPLC Parameters:

The above standards and samples were run on an HPLC column using theparameters shown in Table 20.

TABLE 20 HPLC Parameters Column Temperature: 25° C. AutosamplerTemperature: 37° C. Flow rate: 1.0 mL/min. Flow rate may be adjusted±0.2 mL/min to obtain specified retention times. Time, %-Solvent%-Solvent Gradient: min A* B* Initial 96  4 10 96  4 20 75 25 25 75 25Re-equilibration time 10 minutes Injection volume: 25 μL Needle WashSolution: Use the diluting solution Detection: 205 nm UV Run Time: 25minutes *Solvent A: water; solvent B: acetonitrile

The retention time for decitabine was found to be approximately 8minutes; and the retention time of ER-876437 was found to beapproximately 21.8 minutes.

Results and Discussion

TABLE 21 Summary of Results Solutions Estimated T_(1/2) Decitabine inTris-HCl buffer at 37° C. 9 hours Decitabine with CDA in Tris-HCl bufferat 37° C. 23 minutes Decitabine with CDA and ER-876437-00 in Tris-HCl 9hours buffer at 37° C.

The levels of decitabine, in the presence and absence of CDA, with orwithout ER-876437, in Tris-HCl buffer at 37° C. were measured by HPLCanalysis using UV detection. The areas of the decitabine and ER-876437peaks in the stability samples were measured and compared to the areasof the decitabine and ER-876437 standard controls, respectively. Resultswere reported as percent remaining of control.

Data was collected at 205 nm UV because decitabine and ER-876437 sharethis UV maximum. See FIG. 13. Results were captured every 35 minutes for12 hours and intermittently thereafter due to the length of theanalytical method. HPLC Chromatograms showing overlaid traces atspecified time points are shown in FIGS. 14 and 15.

HPLC chromatograms in these figures are shown with a constant, additiveoffset for clarity. Although the bottom trace is shown starting attime=0.00 minutes, each successive chromatogram is arbitrarily shiftedto the right of the previous chromatogram (by a constant amount of time)so as to avoid having the peaks overlap. The actual times associatedwith the peaks shown in these chromatograms can be realized by shiftingthe start of the chromatogram trace (at the left hand side) back to thevertical axis where time equals 0.00 minutes. Similarly, the actual UVabsorption of any peak can be realized by shifting the baseline of thechromatogram to the position where mAU=0.00.

In the absence of CDA, the reduction of decitabine concentration to 50%was observed after 9 hours, while, in the presence of CDA, theconcentration of decitabine was reduced to nearly 0% control after 2hours and the T₁₁₂ was estimated to be approximately 23 minutes.Addition of ER-876437 to the incubation mixture resulted in inhibitionof the reaction with the reduction of decitabine concentration to 50%observed after 9 hours. The levels of ER-876437 were not affected after12 hours exposure to CDA with decitabine. A summary of all the resultsare shown in FIG. 16.

The estimated T_(1/2) of decitabine in the presence of CDA in Tris-HClbuffer at 37° C. was found to be 23 minutes. ER-876437 nearly completelyinhibited this effect resulting in the same T_(1/2) as decitabine alonein Tris-HCl buffer at 37 C (9 hours).

All publications or patent applications described herein are herebywholly incorporated by reference.

We claim:
 1. A method of inhibiting the activity of cytidine deaminase(CDA) in a subject in need thereof, comprising administering aneffective amount of a compound of formula I:

wherein: one of R₁ and R₂ is F, and the other is selected from H and F;one of R₃ and R₄ is H, and the other is selected from H and OH;where - - - - - - - is a covalent bond or absent, and R₄ is absentwhen - - - - - - - is a covalent bond; or a pharmaceutically acceptablesalt, a C₁₋₆ alkyl ester, or a C₂₋₆ alkenyl ester thereof.
 2. The methodof claim 1, wherein the compound is a compound of formula VIII:

or a pharmaceutically acceptable salt, a C₁₋₆ alkyl ester, or a C₂₋₆alkenyl ester thereof.
 3. A method of inhibiting the deamination of aCDA substrate by CDA in a subject in need thereof, comprisingadministering an effective amount of a compound of formula I:

wherein: one of R₁ and R₂ is F, and the other is selected from H and F;one of R₃ and R₄ is H, and the other is selected from H and OH;where - - - - - - - is a covalent bond or absent, and R₄ is absentwhen - - - - - - - is a covalent bond; or a pharmaceutically acceptablesalt, a C₁₋₆ alkyl ester, or a C₂₋₆ alkenyl ester thereof.
 4. The methodof claim 3, wherein the compound is a compound of formula VIII:

or a pharmaceutically acceptable salt, a C₁₋₆ alkyl ester, or a C₂₋₆alkenyl ester thereof.
 5. The method of claim 3, wherein the CDAsubstrate is decitabine.
 6. The method of claim 3, wherein the CDAsubstrate is a non-decitabine CDA substrate.
 7. The method of claim 6,wherein the CDA substrate is selected from the group consisting ofgemcitabine, 5-azacytidine, ara-C, tezacitabine,5-fluoro-2′-deoxycytidine, and cytochlor.
 8. A method of treating sicklecell anemia in a subject in need thereof, comprising administering tothe subject an effective amount of decitabine and a compound of formulaI:

wherein: one of R₁ and R₂ is F, and the other is selected from H and F;one of R₃ and R₄ is H, and the other is selected from H and OH;where - - - - - - - is a covalent bond or absent, and R₄ is absentwhen - - - - - - - is a covalent bond; or a pharmaceutically acceptablesalt, a C₁₋₆ alkyl ester, or a C₂₋₆ alkenyl ester thereof.
 9. The methodof claim 8, wherein the compound is a compound of formula VIII:

or a pharmaceutically acceptable salt, a C₁₋₆ alkyl ester, or a C₂₋₆alkenyl ester thereof.
 10. A method of treating a cancer being treatedwith a CDA substrate used for treating cancer, comprising: administeringto a subject a CDA substrate used for treating cancer; administering tothe subject a compound of formula I as defined in claim 1 or apharmaceutically acceptable salt, a C₁₋₆ alkyl ester, or a C₂₋₆ alkenylester thereof; and administering to the subject a pharmaceutical agentselected from an anti-emetic agent, an agent that increases appetite, acytotoxic or chemotherapeutic agent, or an agent that relieves pain. 11.The method of claim 10, wherein the compound is a compound of formulaVIII:

or a pharmaceutically acceptable salt, a C₁₋₆ alkyl ester, or a C₂₋₆alkenyl ester thereof.
 12. The method of claim 10, wherein the CDAsubstrate is decitabine.
 13. The method of claim 10, wherein the CDAsubstrate is a non-decitabine CDA substrate.
 14. The method of claim 13,wherein the CDA substrate is selected from the group consisting ofgemcitabine, 5-azacytidine, ara-C, tezacitabine,5-fluoro-2′-deoxycytidine, and cytochlor.
 15. A packaged cancertreatment for treatment of a cancer to be treated with a CDA substrate,the packaged cancer treatment comprising a compound of formula I asdefined in claim 1 or a pharmaceutically acceptable salt, a C₁₋₆ alkylester, or a C₂₋₆ alkenyl ester thereof; and instructions for using aneffective amount of the compound for treating cancer.
 16. The packagedcancer treatment of claim 15, wherein the compound is a compound offormula VIII:

or a pharmaceutically acceptable salt, a C₁₋₆ alkyl ester, or a C₂₋₆alkenyl ester thereof.
 17. The packaged cancer treatment of claim 15,further comprising a CDA substrate.
 18. The packaged cancer treatment ofclaim 17, wherein the CDA substrate is decitabine.
 19. The packagedcancer treatment of claim 17, wherein the CDA substrate is anon-decitabine CDA substrate.
 20. The packaged cancer treatment of claim19, wherein the CDA substrate is selected from the group consisting ofgemcitabine, 5-azacytidine, ara-C, tezacitabine,5-fluoro-2′-deoxycytidine, and cytochlor.
 21. An in vitro method ofinhibiting the activity of CDA, comprising contacting CDA with aneffective amount of a compound of formula I:

wherein: one of R₁ and R₂ is F, and the other is selected from H and F;one of R₃ and R₄ is H, and the other is selected from H and OH;where - - - - - - - is a covalent bond or absent, and R₄ is absentwhen - - - - - - - is a covalent bond; or a pharmaceutically acceptablesalt, a C₁₋₆ alkyl ester, or a C₂₋₆ alkenyl ester thereof.